Use of Epothilone D in Treating Tau-Associated Diseases Including Alzheimer&#39;s Disease

ABSTRACT

Methods of treating Tau-associated diseases, preferably tauopathies, are described using epothilone D that exhibit good brain penetration, long half-life, and high selective retention in brain, and provides effective therapies in treating tauopathies including Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Continuation application claims the benefit of U.S. Ser. No.13/150,671 filed Jun. 1, 2011, now allowed, which in turn is aContinuation application which claims the benefit of Non-Provisionalapplication U.S. Ser. No. 12/429,492 filed Apr. 24, 2009, now abandoned,which in turn claims the benefit of Provisional application U.S. Ser.No. 61/047,729 filed Apr. 24, 2008, now expired.

FIELD OF THE INVENTION

This invention relates generally to the treatment of Tau-associateddiseases using epothilone D, and more specifically, to the treatment ofAlzheimer's Disease using epothilone D.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the most common form of dementia, affectingan estimated 27 million people worldwide in 2006. Age is the greatestknown risk factor for AD with an incidence of 25-50% in people aged 85years or older. As the average age of the population increases, thenumber of patients with AD is expected to rise exponentially. AD is thefifth leading cause of death in people aged 65 and older, and mostpatients eventually need nursing home care. Consequently, AD has anenormous economic impact, e.g., estimated direct and indirect costs for2005 in the US only were $148 billion. Besides the economic costs, ADhas a devastating impact upon patients and their family members, causingsevere emotional distress and turmoil.

Patients are diagnosed with probable AD based on the presence ofdementia with progressive worsening of memory and other cognitivefunctions and with the exclusion of other causes of dementia. Adiagnosis of AD can only be confirmed post-mortem as the clinicaldiagnosis is based on brain neuropathology, specifically, the diagnosisrequires an evaluation of brain tissue, including the existence andconcentration of extracellular plaques in the brain, intracellulartangles, and brain neurodegeneration. Dementia is also a required partof the diagnosis, since plaques and tangles are observed in cognitivelynormal adults, although usually to a lesser extent.

Two classes of medications, cholinesterase inhibitors and anN-methyl-D-aspartic acid (NMDA) antagonist, are currently approved forAD. Although these two classes of therapeutics show some clinicalbenefit, many patients do not respond, and these drugs only amelioratethe symptoms of AD (e.g., cognitive function) with little or nomodification of disease progression. For these reasons, identificationof disease-modifying therapeutics for this devastating disease is amajor focus of the pharmaceutical industry.

Microtubule stabilizers have been suggested as therapies to treattauopathies including AD. See, e.g., Lee et al. (references list,infra). In U.S. Pat. No. 5,580,898, filed May 1994 and granted Dec. 3,1996, Trojanowski et al. suggest use of paclitaxel (TAXOL®) to treat ADpatients by stabilizing microtubules. Paclitaxel has proven highlyeffective as a microtubule-stabilizing agent in treating cancerpatients; however, it presents brain-penetration and peripheralneuropathy issues when considered for AD (further described below), andhas not emerged as a viable therapy to treat AD.

In 1995, epothilone B was reported to exert microtubule-stabilizingeffects similar to paclitaxel (Bollag et al. 1995). Epothilone A andepothilone B are naturally-occurring compounds that were isolated byHofle et al. from fermentation products of the microorganism Sorangiumcellulosum (e.g., WO 93/10121). Hofle et al. also discovered 37 naturalepothilone variants and related compounds produced by S. cellulosum andmodified strains, including epothilones C, D, E, F and other isomers andvariants (e.g., U.S. Pat. No. 6,624,310).

Unique characteristics of the natural epothilones generated muchinterest in their exploration as potential anti-cancer drugs. Now,nearly twenty years have passed since the first discovery of the naturalepothilones A and B. Hundreds of epothilone analogs have been discoveredand described in various patent applications, and abundant literaturehas published under the rubric, “epothilones” (See, e.g., Altmann etal., references list, infra, at 396-423).

The assignee of the current application has developed ixabepilone, asemi-synthetic analog of epothilone B, for treatment of cancer.Ixabepilone has the structural formula:

The chemical name for ixabepilone is(1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy-8,8,10,12,16-pentamethyl-3-[(1E)-1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-17-oxa-4-azabicyclo[14.1.0]heptadecane-5,9-dione.See also U.S. Pat. No. 6,605,599, assigned to the current assignee,Bristol-Myers Squibb Company (BMS). Ixabepilone is amicrotubule-stabilizing agent that has been approved by the FDA fortreatment of metastatic breast cancer and is sold by BMS under thetradename IXEMPRA®. Ixabepilone can be prepared as described in U.S.Pat. No. 6,605,599 or 7,172,884, incorporated herein by reference.

Other natural epothilones and analogs are in advanced clinical trialsfor treatment of cancer including epothilone B (a/k/a patupilone, orEPO-906), in Phase III trials by Novartis Pharma AG, for treatment ofovarian cancer, and sagopilone (or ZK-EPO), a benzothiazolyl-7-propenylsynthetic analog of epothilone B, in Phase II trials by Bayer ScheringAG for treatment of various cancers including tumors of the ovary,breast, lung, prostate and melanoma. In 2007, a Phase II trial withsagopilone was initiated in the US for treatment of brain metastasesfrom breast cancer. Additionally, an epothilone D analog, KOS-1584, hadadvanced to Phase II clinical trials by Kosan Biosciences, Inc. (now awholly-owned subsidiary of BMS) for treatment of non-small-cell lungcancer and solid tumors, and epothilone D had advanced to Phase IIclinical trials for treatment of cancer by Kosan in collaboration withHoffmann-La Roche, Inc.; however, the clinical trials with epothilone Dfor treating cancer were discontinued in 2007. The structure forepothilone D can be represented by the following formula:

The epothilone D compound is claimed, as composition of matter, in U.S.patent application Ser. No. 09/313,524 to Hofle et al., and described inU.S. Pat. Nos. 6,242,469 and 6,284,781 to Danishefsky et al., whichapplication and patents were the subject of Interference No. 105,298,before the USPTO Board of Patent Appeals and Interferences.

The assignee of the current application also has clinically evaluatedBMS-310705 (Compound II herein), for cancer therapy. BMS-310705 waspursued through Phase I clinical trials for treatment of ovarian cancer;it is an amino-epothilone F analog and has the chemical structure:

Compound II (BMS 310705) can be prepared as described in U.S. Pat. No.6,262,094, incorporated herein.

While certain of the epothilone compounds and analogs have beenclinically evaluated for treating cancers, it is highly unpredictablewhether a cancer drug may be effectively used to treat neurodegenerativediseases including AD. There are various factors affecting thisunpredictability. One factor is the substantial difficulty of achievinggood brain penetration due to the blood-brain barrier (BBB). For acompound to be useful in treating neurodegenerative brain diseases, itis necessary that the compound cross the BBB; however, since a functionof the BBB is to protect the brain from external substances and toxins,discovering a useful drug that has good BBB penetration is challenging.Additionally, BBB penetration is an undesirable feature for a cancerdrug (other than brain cancer drugs). With a cancer drug, BBBpenetration is usually sought to be avoided, whereas for a drug designedto treat AD or other neurodegenerative brain diseases, good BBBpenetration is necessary for the compound to be effective. Thus, forexample, while paclitaxel is a highly-successful cancer drug, it has notemerged as a useful therapy to treat brain diseases such as AD, as ithas a low rate of brain penetration through the BBB.

Further factors affecting the unpredictability of evaluating theusefulness of cancer drugs, particularly microtubule-stabilizing drugs,in treating AD and other brain diseases involve the ability of a drug topenetrate the brain, to be retained in the brain for long periods, andto selectively accumulate in the brain relative to peripheral tissues.These parameters can be measured using brain-to plasma ratios, brainhalf-life, and the ratio of the amount of drug retained in the brain ascompared with peripheral tissues (most particularly the liver).Additionally, measuring brain penetration, retention and selective brainaccumulation with microtubule-stabilizers is complex because thesecompounds are typically rapidly cleared from plasma but more slowlycleared from microtubule-containing tissues, making it important to setappropriate time windows for comparisons of plasma and tissue levels.The brain-to-peripheral-tissue ratio is a particularly importantmeasurement given that microtubule-stabilizing agents at certain dosesare highly cytotoxic to peripheral tissues: when microtubule-stabilizingagents, such as paclitaxel, are administered at chemotherapeutic doses,a peripheral neuropathy and other side effects often occur (Postma etal. 1999). These side effects may be tolerable in treating cancerpatients but a different therapeutic window and acceptable side-effectprofile exists in treating patients suffering from AD and other braindiseases.

Yet further challenges involved with looking to cancer drugs forpotential application to neurodegenerative diseases involve the mode ofadministration and the bioavailability and cytotoxicity associatedtherewith.

In WO 2005/075023 A1, published Jan. 30, 2004, to Andrieux et al. ofINSERM, it is suggested that certain epothilones and analogs includingepothilone A, B, C, D, E, and F, and benzothiazolyl and pyridylepothilone B and D analogs may be useful in treating diseases involvinga neuronal connectivity defect, such as schizophrenia or autism.However, Andrieux et al. disclaimed and thereby taught against use ofthese compounds for treating AD, stating that diseases associated withneuronal connectivity defects (i.e., those claimed in that application)“are different from progressive dementing disorders like Alzheimer,which involve neuronal degeneration.”

In WO 03/074053 (053), to Lichtner et al. of Schering AG (published Sep.12, 2003), there is a broad claim to use of a broad genus of epothilonecompounds and synthetic analogs for treating brain cancer and otherbrain diseases, including primary brain tumor, secondary brain tumor,multiple sclerosis, and AD. Lichtner et al. report certain data on fourcompounds, namely, paclitaxel as compared with the compounds namedtherein as compound 1:4,8-dihydroxy-16-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-1-oxa-7-(1-propyl)-5,5,9,13-tetramethyl-cyclohexadec-13-ene-2,6-dione;compound 2:dihydroxy-3-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-10-propyl-8,8,12,16-tetramethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione;and compound 3:7,11-dihydroxy-3-(2-methylbenzothioazol-5-yl)-10-(prop-2-en-1-yl)-8,8,12,16-tetramethyl-4,17-diooxabicyclo[14.1.0]heptadecane-5,9-dione(see WO '053 publication at page 21).

Notably, Lichtner et al. report brain and plasma concentration data forthe above three epothilone analogs, but only for periods of up to 40minutes. Lichtner et al. are not able to report comparative data againstpaclitaxel on brain-to-plasma levels because their paclitaxel brainlevels were below the level of detection, and they do not report datarelating to brain-to-liver ratios, half-life, or brain retention for anyof the compounds (e.g., concentration of drug in brain tissue overextended periods of time).

In view of the foregoing, there remains a need in the art for methods oftreating tauopathies, particularly Alzheimer's disease.

SUMMARY OF THE INVENTION

The present inventors have discovered based on multiple in vivo studiesincluding behavioral and neuropathological studies, that epothilone Dachieves a surprisingly advantageous profile in treating Tau-associateddiseases, including AD. The inventors have discovered that epothilone Dexhibits a remarkable combination of advantageous properties, making thecompound particularly well-suited to treat such diseases. Theseproperties include not only a high level of brain penetration across theBBB, but also a surprisingly long half-life in the brain and asurprisingly high selective retention rate in the brain as compared withdrug levels found in peripheral tissues, most notably, the liver, overextended periods of time. Additionally, the inventors have furtherdiscovered that surprising, therapeutic advantages in treatingTau-associated diseases, particularly, AD, can be achieved with lowdosages of epothilone D, e.g., with dosages that are approximately100-fold less than those administered to achieve chemotherapeuticeffects. Consequently, the inventors have discovered methods that allowfor therapies in treating Tau-associated diseases with epothilone D,particularly treatment of AD, without causing drug-induced side effectsand/or drug-plasma concentration levels that would require use of theepothilone D to be discontinued. Given the low dose as compared withchemotherapeutic treatments, any side effects are greatly reduced ascompared with side effects that are induced upon administration of theepothilones and analogs for treatment of cancer.

The present invention provides methods of treating Tau-associateddiseases including tauopathies, using epothilone D that exhibit asurprisingly advantageous therapeutic profile, and particularly, amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient.

The present invention further provides a pharmaceutical compositioncomprising epothilone D for treating Tau-associated diseases in apatient, wherein the composition exhibits a treatment profile comprisinggood brain penetrance, long half-life in the brain, and selective brainretention (e.g., high brain-to-liver ratio), as defined herein.Preferred embodiments comprise pharmaceutical compositions for treatingtauopathies, particularly, AD, comprising a therapeutically-effectiveamount of epothilone D and a pharmaceutically acceptable carrier.Further embodiments and aspects of the invention are set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the basic design of an experiment on Tg4510 mice usingepothilone D (Compound I).

FIG. 2 shows the results of a Morris water maze (MWM) test of the Tg4510mice at 2.5 months, prior to dosing with epothilone D (Compound I).

FIG. 3 shows the results of a MWM test of the Tg4510 mice at 4.5 months,after 2 months of dosing with epothilone D (Compound I).

FIG. 4 shows probe data 18 hours after 5 days of training in the 4.5month-old Tg4510 mice dosed for 2 months with epothilone D (Compound I).“TQ” stands for target quadrant, “AR” stands for adjacent right, “AL”stands for adjacent left, and “OP” stands for opposite quadrant. Twomeasures of performance, namely % pathlength (A) and number of platformcrossings (B) are described.

FIG. 5 shows neuronal counts in the CA1 and CA3 regions of thehippocampus in Tg4510 mice at 5.5 months following treatment withvehicle, 1 mpk epothilone D (Compound I), and 10 mpk epothilone D(Compound I).

FIG. 6 shows phosphorylated Tau staining of the Tg4510 mice treated withvehicle, 1 mpk epothilone D (Compound I), and 10 mpk epothilone D(Compound I) in the hippocampus. Representative sections from 3 mice pergroup are shown. AT8 positive staining is dark grey and black.

FIGS. 7A-7B show Gallyas silver staining for neurofibrillary tangles inTg4510 mice treated with vehicle, 1 mpk epothilone D (Compound I), or 10mpk epothilone D (Compound I). FIG. 7A shows representative micrographsof cortical staining, where the black silver stain is positive. Lighterbackground staining and some staining of blood vessels were observed innon-transgenic mice. FIG. 7B shows the quantitation of the silver stainin both cortex and hippocampus.

FIGS. 8A-8D show the concentration of Compound II (FIG. 8A), ixabepilone(FIG. 8B), paclitaxel (FIG. 8C) and epothilone D (Compound I) (FIG. 8D)in the plasma, brain, and liver of mice following intravenousadministration at various intervals of up to 24 hours.

FIG. 9 shows the concentration of epothilone D (Compound 1) and CompoundIII (as described in Example 7 herein) in the brain after oraladministration (35 mpk) up to 5 to 24 hours after dosing.

FIG. 10 shows the concentration of epothilone D in the plasma, brain andliver in mice after time intervals up to one week after dosing.

ABBREVIATIONS

The following are abbreviations of various terms used in thisspecification:

-   3R=three repeats-   4R=four repeats-   AD=Alzheimer's disease-   APP=β-amyloid precursor protein-   BBB=blood-brain barrier-   BMS=Bristol-Myers Squibb, Co.-   CHCl₃=chloroform-   CH₂Cl₂=methylene chloride-   DMAP=4-dimethylaminopyridine-   EtOAc=ethyl acetate-   HPLC=high pressure liquid chromatography-   FDA=US Food and Drug Administration-   FTDP-17=frontotemporal dementia with Parkinsonism linked to    chromosome 17-   H, h, hr=hour/hours-   IP=intraperitoneal-   IV=intravenous-   LDA=lithium diisopropylamide-   LLQ=lower limit of quantification-   <LLQ=below LLQ, not detectable-   MAP=microtubule-associated protein-   MeOH=methanol-   min=minutes-   MTs=microtubules-   mpk=milligram per kilogram-   MWM=Morris water maze-   nM=nanomolar-   NQ=not quantifiable due to one or more datapoints<LLQ-   PEG=polyethylene glycol-   PGP=P-glycoprotein-   PO=per os (oral administration)-   PVP=polyvinylpyrrolidone-   RT=room temperature-   SiO₂=silica gel-   TBAF=tetrabutylammoniumfluoride-   TBS=Tris buffered saline-   TEA=triethylamine-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran-   TPGS=d-α-Tocopheryl polythlene glycol 1000 succinate

DETAILED DESCRIPTION OF THE INVENTION Definitions

“About” or “approximately” as used herein means within an acceptablerange of standard deviation for the particular value as determined byone of ordinary skill in the art, considering the measurement inquestion and the instrument used to make the measurement (i.e., thelimitations of the measurement system). For example, “about” can meanwithin one or more standard deviations. As applied to formulations anddosages, “about” can mean a deviation within 10%, more preferably within5%, and even more preferably, within 2%, of the numbers reported.

The terms “at least x” and “x or more”, or “x or greater”, wherein xdenotes a numerical value, are used interchangeably herein as they areintended to have the same meaning.

“Brain penetrance” refers to the ability of a compound to cross the BBB.Because of the rapid peripheral clearance for most microtubulestabilizing agents, it is important to measure brain-to-plasma ratios atrelatively short times post-dosing, e.g., at periods of about of 20 minto 1 h post-dosing, to assess brain penetrance itself. A compound havinggood brain penetrance as defined herein means a compound which at 20 minto 1 h post-dosing will show a brain-to-plasma ratio of 0.5 or greater,more preferably, 0.8 or greater, and most preferably, a ratio of 1 ormore (again, at a time between 20 min and 1 h post-dosing). In assessingwhether a compound or drug satisfies this standard of high brain/plasmaratio (e.g., as recited in the claims herein), in vivo non-human studiesmust be relied upon as human brain tissue cannot be analyzed to assessdrug concentration.

“Cognitive benefits” means that an improvement or lessening in declineof cognitive function for at least one patient in need of treatment isobserved or reported, as characterized by cognition tests, measures ofglobal function, and activities of daily living and behavior. Typically,cognitive benefits are measured with cognition tests designed to measurecognitive decline in a patient or group of patients. Examples of suchtests include cognition tests like ADAS-cog (Alzheimer's diseaseAssessment Scale, cognitive subscale) and the MMSE (Mini-mental stateexam); behavior tests like the NPI (Neuropsyciatric Inventory); dailyliving activity tests like the ADCS-ADL (Alzheimer's Disease CooperativeStudy-Activities of Daily Living); and global function tests such as theCIBIC-plus (Clinician Interview Based Impression of Change), and CDR sumof boxes (Clinical Dementia Rating).

“Extended periods of time” as used herein means period of 24 hours ormore, typically 24 to 76 h.

“High selective retention rate” or “high selective retention” as usedherein means that the drug or compound is retained in one tissue ororgan, specifically the brain, at a much higher level than is found inother tissues and organs, especially the liver, as measured at anextended period of time post-dosing. More particularly as definedherein, a high selective retention rate means the concentration of drugin the brain is 4 or more times that found in the liver at 24 or more hpost-dosing, more preferably, a factor of at least 6 or more, and mostpreferably, at a factor of at least 8 or more at 24 h or more hpost-dosing. In assessing whether a compound or drug satisfies thisstandard of high selective retention (e.g., as recited in the claimsherein), naturally non-human studies must be relied upon as human braintissue cannot be analyzed to assess drug concentration.

“Impact on underlying disease” means an improvement in a measure of thebiomarkers and other parameters associated with the disease process,including biochemical markers in CSF or plasma, changes in brain volume,changes in brain function as measured by functional imaging, and changesin histopathology or biochemistry that might be observed after autopsy.Typical biomarkers that may be used for AD clinical trials includeanalytes measured in CSF such as Tau, phosphoTau, beta-amyloid, andisoprostanes, as well as brain imaging modalities such asfluorodeoxyglucose PET and volumeteric MRI. Additional biomarkers thatpotentially may be useful, particularly those examining synapticactivity, MT integrity/function, and oxidative stress include, but arenot limited to: GABA, neuropeptide Y, alpha-synuclein, neurogranin andvasoactive intestinal peptide, tubulin, Tau fragments, ubiquitinatedproteins, soluble forms of amyloid precursor protein, chromogranin B,4-hydroxy nonenal, nitrotyrosine, and 8-hydroxy-deoxyguanidine.

“Intermittent” when used with reference to a dosing schedule means thatthere are breaks in the dosing schedule that are irregular. For example,a daily, weekly, biweekly, or monthly dosing schedule is not consideredintermittent under this definition, because the break between doses isin each instance regular and defined by the dose cycle of administeringthe drug. However, a more elaborate dosing schedule with one or moreirregular breaks would be considered intermittent, such as 5 days on,followed by 2 days off; or a dose administered on days 1, 8 and 15, of a30 day cycle, and so forth.

“Long half-life”, or “long brain half-life” as used herein means that adrug has a half-life of 20 or more h post-dosing (which is considereddose-independent), and more preferably, for a period 30 or more hpost-dosing, and most preferably, for a period of 40 or more hpost-dosing. As with the selective retention rates, in vivo non-humanstudies must be relied upon in assessing whether the compound has a longbrain half-life.

“Low dose” as used herein means a dose of the epothilone D compound thatis significantly less than that administered to achieve chemotherapeuticeffects (e.g., given a particular mode of administration, clinicaltrial, and/or experiment), preferably a dose that is 10-fold or lessthan the chemotherapeutic dose, more preferably a dose that is 50-foldor more less, and even more preferably a dose that is 100-fold or morefold less than the chemotherapeutic dose, i.e., that previously assessedas chemotherapeutically effective using the same administrative methodfor the given experiment, study or trial. For example, in Phase IIclinical trials of epothilone D, a dose administered was 100 mg/m²administered as a 90 min. infusion once a week for three weeks everyfour weeks (3 weeks on, 1 week off), for a cumulative total of 300 mg/m²administered every 4 weeks. A low dose relative to this clinical trialdose, as defined herein, would mean a cumulative one-month dosefollowing IV administration of 30 mg/m² or less, more preferably a doseof 6 mg/m² or less, and even more preferably a dose of 3 mg/m² or less.Thus, as an alternative example, a low dose as compared with the aboveclinical trial dose when administered once every 4 weeks would be a doseof 30 mg/m², more preferably a dose of 6 mg/m², and even more preferablya dose of 3 mg/m². Since bioavailability may change depending upon themode of administration (e.g., oral v. IV administration, with lessbioavailability achieved upon oral administration), the relative dosages(i.e., assessment whether a given dose is a “low dose” as definedherein), should be based on a comparison involving the same or similarmodes of administration.

“Patient in need of treatment” as used herein is intended to include useof epothilone D for a patient 1) already diagnosed with a Tau-associateddisease (including a tauopathy, particularly AD) at any clinical stage,including patients having mild cognitive impairment to advanceddementia; and/or 2) who has early or prodromal symptoms and signs of aTau-associated disease (including a tauopathy, particularly AD); and/or3) who has been diagnosed as susceptible to a Tau-associated disease(including a tauopathy, particularly AD), due to age, hereditary, orother factors for whom a course of treatment is medically recommended todelay the onset or evolution or aggravation or deterioration of thesymptoms or signs of disease.

“Statistically significant cognitive benefits” means that there arecognitive benefits (e.g., improvement or the lessening in decline ofcognitive function), following a period of 6 months to a year oftreatment for at least 10% or more of patients evaluated, morepreferably at least 25% or more patients, and even more preferably, 50%or more of the patient group. Preferably, improvement at a rate ascompared with a control group is assessed and reflects an at least 10%improvement (e.g., as evaluated based on comparative test scores betweenplacebo and control, wherein “improvement” is intended to includereduction in decline in a patient's condition), more preferably,improvement at a rate of more than 25% or more is observed, and mostpreferably, at a rate of 35% or more.

“Tau-associated disease” as defined herein means diseases associatedwith abnormalities in Tau as well as diseases that are “tauopathies.”Tau-associated diseases include, but are not limited to, frontotemporaldementia, including the subtype of frontotemporal dementia andParkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclearpalsy, corticobasal degeneration, Pick's disease, agyrophilic graindisease, as well as Parkinson's disease, Down syndrome, post-encephalicParkinsonism, myotonic dystrophy, Niemann-Pick C disease, dementiapugilistica, Blint disease, prion diseases, amyotrophic lateralsclerosis, Parkinsonism-dementia complex of Guam, multiple sclerosis,glaucoma, diabetic retinopathy, and traumatic brain injury; as well asHuntington's disease, Lewy body dementia, Charcot-Marie-Tooth disease,hereditary spastic paraplegia, and multiple system atrophy.

“Tauopathy” as defined herein means a neurodegenerative diseaseassociated with fibrillar forms of Tau protein (tangles) in brain. Thesediseases include AD; however, other tauopathies include, but are notlimited to, frontotemporal dementia, including the subtype offrontotemporal dementia and Parkinsonism linked to chromosome 17(FTDP-17), progressive supranuclear palsy, corticobasal degeneration,Pick's disease, and agyrophilic grain disease.

“Therapeutically-effective amount of epothilone D” is meant an amount ofepothilone D sufficient to:

(1) relieve or alleviate at least one symptom of a Tau-associateddisease (preferably, a tauopathy, and more preferably, AD), includingcognitive functions such as dementia, memory loss, reducedcomprehension, dexterity in performing daily living activities, and/orcentrally-mediated effects such as motor deficits and vision; and/or(2) reverse, reduce, prevent, inhibit, or delay the onset or aggravationof the loss of cognitive function associated with a Tau-associateddisease (preferably, a tauopathy, and more preferably, AD), and/orreverse, reduce, prevent, inhibit, or delay the onset or aggravation ofone or more centrally mediated effects of said disease, including motordeficits, vision, and so on. In preferred embodiments of the invention,the epothilone D pharmaceutical compound is therapeutically effective innot only relieving or alleviating the symptoms of the Tau-associateddisease (preferably, a tauopathy, and more preferably, AD), but also iseffective in having an impact on underlying disease (i.e., as definedabove).

Alternative Embodiments of the Invention

The present inventors have found that epothilone D, administered for thetreatment of a Tau-associated disease achieves a surprising level ofbrain penetration, long brain half-life, and selective retention,particularly as compared with other microtubule stabilizers. Theinventors further have discovered that remarkably, increased therapeuticeffects in treating Tau-associated diseases (particularly tauopathies,and more particularly, AD), are achieved with low doses of epothilone D.As such, a relatively low dosage of epothilone D can be administered foreffective treatment of a Tau-associated disease, preferably AD. Theinventors have thus developed a method of treating Alzheimer's diseaseemploying the administration of epothilone D to a patient having AD. Themethod is expected to be therapeutically effective in treating AD inhuman patients while also posing significantly less serious or fewerside effects as compared with the side effects that typically occur whenmicrotubule stabilizers are administered to human patients forchemotherapy. Such side effects that are reduced or eliminated mayinclude one or more of gastrointestinal distress (including, withoutlimitation, nausea, diarrhea, stomatitis/mucositis, vomiting, anorexia,constipation, and/or abdominal pain), liver toxicity, neutropenia,leucopenia, myelosuppression, alopecia, myalgia/arthralgia, fatigue,musculoskeletal pain, nail disorder, pyrexia, headache, skinexfoliation, and/or neurosensory effects at various grade levels.

According to an alternative embodiment of the invention, there isprovided a method of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D compound has two or more propertiesselected from good brain penetrance, a long brain half-life, and a highselective retention rate, as defined herein, more preferably, where theepothilone D demonstrates all three properties of good brain penetrance,long brain half-life, and a selective retention rate, as these terms aredefined herein.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D compound upon administration hasproperties selected from two or more of:

-   -   brain penetrance of 0.5 or greater, more preferably, 0.8 or        greater, most preferably, 1 or greater, as measured at 20 min.        to 1 h post-dosing; and/or    -   a brain half-life of at least 24 h, and more preferably, of at        least 30 h, and most preferably of up to 40 h or more; and/or    -   a brain-liver selective retention rate of at least 4 at 24 h or        more post-dosing, more preferably at a rate of 6 or more at 24 h        or more, and most preferably, at a factor of 8 or more at 24 or        more h post-dosing.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D compound upon administration hasproperties selected from all three of:

-   -   brain penetrance of 0.5 or greater, more preferably, 0.8 or        greater, most preferably, 1 or greater, as measured at 20 min.        to 1 h post-dosing; and/or    -   a brain half-life of at least 24 h, and more preferably, of at        least 30 h, and most preferably of up to 40 h or more; and/or    -   a brain-liver selective retention rate of at least 4 at 24 h or        more post-dosing, more preferably, at a rate of 6 or more at 24        h or more, and most preferably at a factor of 8 or more at 24 or        more h post-dosing.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the method is therapeutically effective in treating ADin the patient without causing drug-induced side effects and/ordrug-plasma concentration levels that would require use of said methodto be discontinued.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the method provides cognitive benefits, morepreferably, statistically-significant cognitive benefits, in treatingAD, without causing drug-induced side effects and/or drug-plasmaconcentration levels that would require use of said method to bediscontinued.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the method has an impact on underlying disease, morepreferably, a statistically-significant impact on underlying disease,without causing drug-induced side effects and/or drug-plasmaconcentration levels that would require use of said method to bediscontinued.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein method has an impact on underlying diseases, providescognitive benefits, and/or is otherwise therapeutically effective,without causing side effects such as gastrointestinal side effects,leucopenia, and/or neurotoxicity, that would require use of said methodto be discontinued.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the dose of epothilone D is a low dose, as definedherein.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the dose of epothilone D is between 0.001-10 mg/m², oralternatively, at a dose between 0.00003-0.3 mpk, administered on adaily, weekly, or intermittent dosing cycle.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D is administered via IV, and the doseof epothilone D over a cumulative monthly dosing cycle (i.e., totaldosage of compound administered over a one month cycle, regardless ofschedule, e.g., weekly, bi-weekly, 3 week on, 1 week off, etc.) is inthe range between 0.001-5 mg/m², more preferably between 0.01-5 mg/m²,even more preferably between 0.01-3 mg/m², yet even more preferablybetween 0.1-3 mg/m², and most preferably between 0.1-1 mg/m².

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D is administered orally, and the doseof epothilone D calculated on a daily basis is in the range between0.001-2 mg/m², more preferably between 0.01-2 mg/m², even morepreferably between 0.1-2 mg/m², yet even more preferably between 0.2-2mg/m².

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D is administered orally, and the doseof epothilone D for a cumulative monthly basis (i.e., total dosage ofcompound administered over a one month cycle, regardless of schedule,e.g., daily, weekly, bi-weekly, etc.) is in the range between 0.03-60mg/m², more preferably between 0.30-60 mg/m², even more preferablybetween 3-60 mg/m², yet even more preferably between 6-60 mg/m².

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D is administered orally on a dosingschedule selected from once daily, once weekly, once every two weeks, oronce a month.

According to another embodiment of the invention, there is provided amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the epothilone D is administered orally on a dosingschedule selected from once daily, and wherein the daily dose ofepothilone D is between 0.2 to 2 mg/m².

According to another aspect of the invention, there are provided methodsof treating other tauopathies, besides AD, according to any one of theembodiments of the invention recited above. For example, such othertauopathies may include one or more of the diseases referenced in thedefinition of “tauopathy-associated disease” herein. For example, oneembodiment of the invention comprises use of epothilone D, according toany of the above embodiments, to treat not only AD but also a diseaseselected from frontotemporal dementia, including the subtype offrontotemporal dementia and Parkinsonism linked to chromosome 17(FTDP-17), progressive supranuclear palsy, corticobasal degeneration,Pick's disease, and agyrophilic grain disease, Parkinson's disease, Downsyndrome, post-encephalic Parkinsonism, myotonic dystrophy, Niemann-PickC disease, dementia pugilistica, Blint disease, prion diseases,amyotrophic lateral sclerosis, Parkinsonism-dementia complex of Guam,multiple sclerosis, glaucoma, diabetic retinopathy and/or traumaticbrain injury. A preferred embodiment comprises use of epothilone D,according to any of the embodiments described herein, to treat atauopathy, including, without limitation, a disease selected from AD,frontotemporal dementia, including the subtype of frontotemporaldementia and Parkinsonism linked to chromosome 17 (FTDP-17), progressivesupranuclear palsy, corticobasal degeneration, Pick's disease, andagyrophilic grain disease.

According to another embodiment of the invention, there is providedepothilone D, for use in treating a Tau-associated disease, morepreferably, a tauopathy, most preferably AD.

It is contemplated that each of the above inventive methods also may becombined with one or more other inventive methods, and all such variouscombinations of the above inventive methods are contemplated herein. Forexample, one combination of the above inventive methods may comprise amethod of treating Alzheimer's disease comprising the step ofadministering a therapeutically effective amount of epothilone D to apatient, wherein the method is therapeutically effective in treating ADin the patient without causing drug-induced side effects and/ordrug-plasma concentration levels that would require use of said methodto be discontinued; and wherein the dose of epothilone D is a cumulativemonthly dose of between 0.001-5 mg/m², administered via IV; and/orwherein the dose of epothilone D is between 0.001 to 2 mg/m²,administered PO daily; and/or wherein the dose of epothilone D isselected from a dose within any one of the preferred ranges expressedabove for oral or IV administration.

It also is contemplated also that any of the recited methods oftreatment may by combined with the embodiment involving epothilone D,for use in treating a tauopathy, preferably AD, in a human patient.Thus, for example, one embodiment of the invention, comprising acombination of the above alternative embodiments, would compriseepothilone D for treating AD, wherein the use is therapeuticallyeffective in treating AD, and wherein the epothilone D is administeredto the patient at a dose between 0.001-10 mg/m², or alternatively, at adose between 0.00003-0.3 mpk, administered on a daily, weekly, orintermittent dosing cycle. Yet another embodiment would compriseepothilone D, for treating a tauopathy, particularly AD, wherein theepothilone D is administered to a human patient at a low dose and istherapeutically effective in having an impact on underlying diseaseand/or providing cognitive benefits.

According to another embodiment of the invention, there is provided apharmaceutical formulation comprising epothilone D suitable foradministration to a human patient in need of treatment for aTau-associated disease, preferably a tauopathy, more preferably, AD,wherein administration of the formulation is therapeutically effectivein treating the disease in the patient without causing drug-induced sideeffects and/or drug-plasma concentration levels that would require useof said epothilone D formulation to be discontinued.

According to yet another embodiment of the invention, there is provideda pharmaceutical formulation comprising epothilone D suitable foradministration to a human patient for treating a Tau-associated disease,preferably a tauopathy, more preferably AD, wherein administration ofthe formulation provides statistically-significant cognitive benefits intreating the disease, without causing drug-induced side effects and/ordrug-plasma concentration levels that would require use of saidepothilone D formulation to be discontinued.

According to yet another embodiment of the invention, there is provideda pharmaceutical formulation comprising epothilone D suitable foradministration to a human patient for treating a Tau-associated disease,preferably a tauopathy, more preferably, AD, wherein the formulation iseffective in providing an impact on underlying disease, without causingdrug-induced side effects and/or drug-plasma concentration levels thatwould require use of said epothilone D formulation to be discontinued.

According to yet another embodiment of the invention, there is provideda pharmaceutical formulation suitable for administration to a humanpatient for treating a Tau-associated disease, preferably a tauopathy,more preferably, AD, wherein the formulation comprises a dosage unit ofepothilone D of between 0.0001-10 mg/m², more preferably between 0.001-5mg/m², more preferably between 0.001-3 mg/m², even more preferablybetween 0.001-1 mg/m², and most preferably between 0.001-0.5 mg/m².

According to yet another embodiment of the invention, there is provideda pharmaceutical formulation for IV administration to a human patient,wherein said formulation is suitable for delivery of a cumulativemonthly dose of epothilone D in the range between 0.001-5 mg/m², morepreferably between 0.01-5 mg/m², even more preferably between 0.01-3mg/m², yet even more preferably between 0.1-3 mg/m², and most preferablybetween 0.1-1 mg/m².

According to yet another embodiment of the invention, there is provideda pharmaceutical formulation for oral administration to a human patient,wherein said formulation is suitable for delivery of a cumulativemonthly oral dose of epothilone D in the range between 0.03-60 mg/m²,more preferably between 0.30-60 mg/m², even more preferably between 3-60mg/m², yet even more preferably between 6-60 mg/m².

According to yet another embodiment of the invention, there is provideda pharmaceutical formulation for administration to a human patient,wherein said formulation comprises epothilone D in a pharmaceuticallyacceptable solvent system comprising from about 0 to 50% propyleneglycol, about 1 to 10% TPGS, about 0.5 to 10% ethanol, about 0-90%water, and/or about 5 to 85% PEG such as PEG-400.

Combinations of each of the above inventive pharmaceutical formulationsare also contemplated herein.

Utility Tauopathies

Tauopathies are neurodegenerative diseases associated with abnormalforms of Tau protein in brain tissue. Alzheimer's Disease (AD) was thefirst neurodegenerative disease to be identified as implicating Taudysfunction. In particular, neurofibrillary tangles—the presence ofwhich is one of the hallmark pathologies in AD—were found to containfibrillar, hyperphosphorylated, conformationally-altered forms of theTau protein. Subsequently, other tauopathies were identified includingfrontotemporal dementia and Parkinsonism linked to chromosome 17(FTDP-17), progressive supranuclear palsy, corticobasal degeneration,Pick's disease, and agyrophilic grain disease. In addition, a link withTau abnormalities (including hyperphosphorylated Tau, Tau aggregates,and/or an association with the H1/H1 Tau haplotype) has been associatedwith Parkinson's disease, Down syndrome, post-encephalic Parkinsonism,myotonic dystrophy, Niemann-Pick C disease, dementia pugilistica, Blintdisease, prion diseases, amyotrophic lateral sclerosis,Parkinsonism-dementia complex of Guam, multiple sclerosis, glaucoma,diabetic retinopathy and traumatic brain injury (Avila et al. 2004;Bartosik-Psujek et al. 2006; Dickey et al. 2006; Wostyn et al. 2008).

Tau is a 50 to 75 kDa microtubule-associated protein (MAP) that bindsand stabilizes microtubules (MTs). There are six primary sequencevariants of Tau, and these variants are formed by alternative splicing(Lace et al. 2007). The splice variants contain zero, one, or two (0N,1N, or 2N) N-terminal inserts in combination with either three repeats(3R) or four repeats (4R) of a microtubule-binding domain. The repeatdomains are necessary for microtubule stabilization, while proline-richregions on either side of the repeat domains are necessary for bindingto the microtubules (Preuss et al. 1997). The repeat domains andproline-rich regions are phosphorylated by multiple kinases, leading todissociation of Tau from microtubules. The N-terminus of Tau extendsaway from the microtubule surface, where it is believed to assist indetermining the spacing between microtubules and in binding of the motorprotein dynactin to the microtubules (Magnani et al. 2007). In normalcells, there are roughly equal levels of 3R Tau and 4R Tau present. 4RTau binds more tightly to microtubules than does 3R Tau.

Tau is most abundant in neurons where it is predominantly localized toaxons. Tau is the major microtubule-associated protein in neuronalaxons, while the MAP1 family is widely distributed in neurons, and theMAP2 family is predominantly somatodendritic. Tau stabilizes axonalmicrotubules, thereby facilitating transport of proteins, organelles,lipids, cellular components targeted for degradation, and cell signalingmolecules bi-directionally between the cell body and the synapticterminals. Tau dysfunction could interfere with axonal trafficking andthereby affect neuronal function and survival. The Tau gene can beknocked out in mice with mild consequences, but if both Tau and MAP 1Bgenes are removed, the double knockout mice die as embryos. It appearsthat alterations in expression of some MAPs can substitute for eachother in many cases, including development (Avila et al. 2004).

In some cases, mutations in the gene encoding Tau (sometimes calledMAPT) cause tauopathies, particularly in FTDP-17 and otherfrontotemporal dementias. Many FTDP-17 mutations decrease binding tomicrotubules in vitro and/or increase their propensity to form fibrils(Lace et al. 2007). Other tauopathy-associated mutations alter thesplice pattern of Tau to generate predominantly 3R or 4R Tau. Yetanother class of Tau mutations on the N-terminus alters the ability tobind to dynactin (Magnani et al. 2007). All of these mutations have thepotential to interfere with normal functions of Tau. In the case of AD,it is thought that β-amyloid (Aβ) leads to abnormalities in Tau.

Although tangles and other Tau aggregates are a pathologic feature oftauopathies, several lines of evidence suggest that some other, soluble,unidentified form of abnormal Tau is neurotoxic. The data suggestingthat Tau aggregated into neurofibrillary tangles is not directlypathogenic include observations of human brains and mouse models. Forinstance, examination of different regions and disease stages ofAlzheimer's disease brains has led to the conclusion that neurons cansurvive and function with neurofibrillary tangles for decades (Morsch etal. 1999). Likewise, human Tau (hTau) transgenic mice have tangles andsevere neurodegeneration, but the neurons with tangles do not showselective signs of distress and are too few in number to account for thedramatic loss in neurons observed in this model (Andorfer et al. 2005).Tg4510, an inducible Tau transgenic line, shows dramatic and rapidtangle formation, neurodegeneration, and behavioral deficits whenTau-P301L is induced (Santacruz et al., 2005). When Tau-P301L expressionis repressed, neurodegeneration and cognitive deficits are greatlyreduced, but tangle formation continues. Further studies using thesemice show that soluble Tau multimers correlate with cognitive deficits.Similar Tau multimers are also observed in FTDP-17 and AD brain tissue(Berger et al. 2007). Finally, evidence for non-fibrillar Tau beinginvolved in behavioral deficits in AD was obtained using transgenic miceoverexpressing a mutant form of β-amyloid precursor protein (APP)(Roberson et al., 2007). When these APP mice were crossed with Tauknockout mice, amyloid plaques were formed, but behavioral deficits andsynaptic abnormalities were prevented. In this APP line, Tauabnormalities could not be detected in the presence of synaptic andbehavioral deficits. Taken together, these studies show that a soluble,unidentified form of abnormal Tau is likely the neurotoxic species.

Microtubule Stabilization for Treatment of Tauopathies

There are two major hypotheses for the role of Tau in neurodegenerativedisease. One hypothesis posits that abnormal forms of Tau disruptcellular function, while the other hypothesis posits that the loss offunctional Tau leads to microtubule destabilization (Avila et al. 2004;Lace et al. 2007). It is based on the second hypothesis that microtubulestabilizers have been suggested as therapies to treat tauopathies (Leeet al. 1994; U.S. Pat. No. 5,580,898). Inappropriate disruptions inaxonal trafficking have been implicated in a number of diseases inaddition to those identified with abnormalities in Tau. These includeHuntington's disease, Lewy body Dementia, Charcot-Marie-Tooth disease,hereditary spastic paraplegia, and multiple system atrophy (Roy et al.2005).

To test if microtubule stabilizers could benefit mice that overexpressTau, PrP T44 Tau transgenic mice were treated with paclitaxel (Zhang etal., 2005). PrP T44 mice overexpress normal human 0N3R Tau in spinalcord neurons and consequently develop motor deficits due to Tauoverexpression. Paclitaxel treatment for 3 months reduced motordysfunction and increased microtubule numbers and axonal transport inthe ventral roots of the spinal cord. Although paclitaxel is poorlyCNS-penetrant, it was able to influence the efferent axons from neuronsin the ventral horn of the spinal cord which are outside the blood-brainbarrier. Interestingly, Tau pathology in this model (spheroids) wasunaffected. Since this model does not show neuronal loss, the effect ofmicrotubule stabilizers on neuronal survival could not be assessed.Additionally, it is unclear whether the motor benefits observed in thespinal cord tauopathy would translate into cognitive benefits in acortical-hippocampal tauopathy. For example, opposite results wereobserved with the cross of two different tauopathy transgenic mouselines to transgenic mice that overexpress glycogen synthase kinase 3(Gsk3). In the spinal cord tauopathy model, the Tau-Gsk3 bigenic animalshad reduced pathology, while in the forebrain tauopathy model, theTau-Gsk3 bigenic animals showed increased pathology (Spittaels et al.2000; Terwel et al. 2008).

Microtubule stabilization has been offered as an explanation for theeffects of NAP, a peptide of sequence NAPVSIPQ, in several animalmodels. In particular, NAP has neurotrophic, anti-inflammatory,anti-apoptotic, and neuroprotective activities in many cellular and invivo models, including middle cerebral artery occlusion (stroke model),head trauma, cholinotoxic lesions, aging, and developmental defects infetal alcohol syndrome and apolipoprotein E deficient mice (Gozes et al.2006; Gozes 2007). As for tauopathies, NAP administration for 3 or 6months is reported to reduce AD levels, hyperphosphorylated Tau, andsarcosyl insoluble Tau while increasing soluble Tau in 3×Tg mice(Matsuoka et al. 2007; Matsuoka et al. 2008). 3×Tg mice overexpress APPand Tau-P301L (Oddo et al. 2003). The mechanism of NAP activity is notfully defined, but there is evidence, based on binding of tubulin to aNAP affinity column and effects on microtubule formation and/orstabilization in cultured neurons, that NAP binds to microtubules(Divinski et al. 2006). NAP is also known to inhibit AD aggregation, soit may be acting upstream of Tau in the 3×Tg model. NAP is not likely toact as a typical microtubule-stabilizing agent, as it is able to protectagainst paclitaxel-induced peripheral neuropathy in rats (U.S. PatentApplication Publication No. 2006/0247168 A1). Whenmicrotubule-stabilizing agents, such as paclitaxel, are administered athigh, chemotherapeutic doses, a peripheral neuropathy often occurs(Postma et al. 1999) that is believed to result from theover-stabilization and bundling of microtubules in peripheral nerves.Since NAP prevents paclitaxel-induced peripheral neuropathy in rats,paclitaxel and NAP are unlikely to act through identical mechanisms.

There are also suggestions that microtubule stabilizers could haveneuroprotective effects unrelated to obvious Tau dysfunction.Microtubule-stabilizing compounds protect cultured neurons from multipletoxic insults, including Aβ42, oxidative stress from soluble Aβ40,lysosomal disruption, calcium-induced toxicity, and glutamate-inducedtoxicity (Burke et al. 1994; Furukawa 1995; Sponne et al. 2003;Michaelis et al. 2005; Butler et al. 2007). It is hypothesized thatmicrotubules play a key role not only in transport mechanisms, but alsoin regulation of cell signaling, particularly calcium signaling,possibly through anchoring of macromolecular signaling complexes in thevicinity of the plasma membrane (Michaelis et al. 2005). Microtubulestabilizing agents have also been shown to enhance mitochondrialfunction by reducing reactive oxygen species generation and increasingexpression of the oxidative phosphorylation genes involved in ATPproduction (Wagner et al. 2008). Microtubule-stabilizing agents are alsoknown to broadly influence cell signaling during disruption of themitotic spindle in cancer cells (Bergstralh et al. 2006).

Brain-Penetrant Microtubule Stabilizers

The therapeutic target of microtubule stabilizers for tauopathies andother neurodegenerative diseases is microtubules in the brain. However,microtubule stabilizers can cause toxicity to peripheral tissues, suchas inhibition of cell proliferation, particularly in thegastrointestinal tract and hematopoietic cells, and peripheralneuropathy. It is thus highly desired to identify microtubulestabilizers with excellent brain penetration and selective retention inthe brain as compared with peripheral tissues, so as to maximize thetherapeutic index for tauopathies and other neurodegenerative diseases.The ability of compounds to bind with a longer half life to brain tissuerelative to peripheral tissues is a highly desired property.

The taxane series of microtubule stabilizers are substrates of multiplemulti-drug resistance transporters, such as P-glycoprotein (PGP),ATP-binding cassette, multidrug resistance protein, and breast cancerresistance protein. These multi-drug resistance transporters preventcompounds from accumulating in tumor and brain tissue. Multiple labshave worked to synthesize taxanes that are not substrates for multi-drugresistance transporters, particularly PGP, with limited success(Minderman et al. 2004; Rice et al. 2005; Ballatore et al. 2007).Co-administration of a PGP inhibitor with paclitaxel has also beenattempted (Fellner et al. 2002). These efforts have shown results ofsome taxane entry into the brain, achieving, for example, approximately1/30th the levels of paclitaxel in the brain as in the kidney with a PGPinhibitor, or brain levels in the μM range with KU-237, but only for 4 hafter administration (Michaelis 2006). Hence, use of PGP inhibitors arenot an attractive method to increase the brain penetration of taxanes.

Methods of Preparation and Formulations

Epothilone D is a known compound which has been chemically synthesizedde novo and also has been isolated from fermentations of Sorangiumcellulosum strains as minor products in the fermentation of S.cellulosum. Total synthesis of epothilone D is reported in U.S. Pat. No.6,242,469 to Danishefsky et al., and additional methods for preparingepothilone D and other epothilone compounds can be found at U.S. Pat.Nos. 6,204,388, 6,288,237, 6,303,342;

WO 03/072730, U.S. Pat. No. 6,410,301; U.S. Patent ApplicationPublication No. 2002/0137152A1; U.S. Pat. No. 6,867,333, U.S. PatentApplication Publication No. 2006/004065, each of which is incorporatedherein by reference. Synthetic methods for manufacturing epothilone Dhave been characterized as impractical for full-scale pharmaceuticaldevelopment. One alternative method of preparation is to engage inlarge-scale fermentation of epothilone B, for example, as described inU.S. Pat. No. 7,172,884 B2, with use of improved strains designed toprovide relatively large yields of epothilone B, and the epothilone Bcan be de-epoxidized to provide epothilone D. Methods of de-epoxidationare well known but also can be found in U.S. Pat. No. 6,965,034 (WO99/43653), to Danishefsky et al., particularly as applied to epothiloneD.

Further methods for making epothilone D are set forth in U.S. Pat. Nos.6,998,256 B2 and 7,067,286, “Methods of Obtaining Epothilone D usingCrystallization and/or By the Culture of Cells in the Presence of MethylOleate,” which describe the biosynthetic production of epothilone Dusing Myxococcus xanthus strains K111-40-1 and K111-72.4.4, and/or otherrecombinant strains that have been developed by Kosan Biosciences Inc.(now BMS), to improve production of epothilone D. Fermentation andpurification conditions for making epothilone D are also set forth inU.S. Pat. Nos. 6,998,256 B2 and 7,067,286, as well as U.S. Pat. Nos.6,583,290, 6,858,411, 6,921,650, and 7,129,071, each of which isassigned to Kosan (now BMS, the current assignee), and incorporatedherein by reference. See also, Lau et al., Kosan Biosciences,“Optimizing the Heterologous Production of Epothilone D in Myxococcusxanthus,” Biotechnology & Bioengineering, 78(3):280-288 (May 5, 2002).

Yet further methods that may be used in making epothilone D areillustrated in U.S. patent application Ser. No. 12/118,432. Thisapplication discloses a combination of chemical and biosynthetic stepsto prepare epothilones such as epothilone D. For example, methods areprovided in which one or more intermediates that may be used forepothilone synthesis are obtained through fermentation of recombinantcells, and then the biosynthesized intermediates with use of recombinantcells, disclosed therein, are converted to the final epothilonecompounds via chemical synthesis.

The epothilone D used in methods of the present invention can beadministered to a patient in various ways known in the art, typically byintravenous (IV) administration, subcutaneous administration, oraladministration, and so on. For example, epothilone D can be formulatedwith a pharmaceutically acceptable vehicle or diluent. A pharmaceuticalcomposition comprising epothilone D can be formulated in a classicalmanner using solid or liquid vehicles, diluents, and additivesappropriate to the desired mode of administration.

Exemplary compositions for parenteral administration include injectablesolutions or suspensions which can contain, for example, suitablenon-toxic, parenterally acceptable diluents or solvents, such asmannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodiumchloride solution (0.9% Sodium Chloride Injection [Normal Saline] or 5%Dextrose Injection), or other suitable dispersing or wetting andsuspending agents, including synthetic mono- or diglycerides, and fattyacids. Pharmaceutically acceptable compositions and/or methods ofadministering compounds of the invention may include use of co-solventsincluding, but not limited to ethanol, N,N dimethylacetamide, propyleneglycol, glycerol and polyethylene glycols, e.g., polyethylene glycol 300and/or polyethylene glycol 400. Surfactants (pharmaceutically-acceptablesurface active agent) may be used to increase a compound's spreading orwetting properties by reducing its surface tension, including withoutlimitation, d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS),Cremophor, Solutol HS 15, polysorbate 80, polysorbate 20, poloxamer,pyrrolidones such as N-alkylpyrrolidone (e.g., N-methylpyrrolidone)and/or polyvinylpyrrolidone; however, use of Cremophor has disadvantagesand is not preferred. The formulation may also comprise use of one ormore “buffers” (e.g., an ingredient which imparts an ability to resistchange in the effective acidity or alkalinity of a medium upon theaddition of increments of an acid or base), including, withoutlimitation, sodium phosphate, sodium citrate, diethanolamine,triethanolamine, L-arginine, L-lysine, L-histidine, L-alanine, glycine,sodium carbonate, tromethamine (a/k/a tris[hydroxymethyl]aminomethane orTris), and/or mixtures thereof.

Formulations for administering epothilone compounds, includingformulations that avoid use of non-ionic surfactants such as Cremophor,are described in the prior art. For example, a formulation for use in IVadministration that comprises a mixture of propylene glycol and ethanolis described in U.S. Pat. No. 6,683,100. Further formulations maycomprise mixtures of polyethylene glycol/dehydrated alcohol, orpropylene glycol or glycerol/dehydrated alcohol. For example, WO2006/105399 (PCT/US2006/011920) to BMS, discloses formulations thatinclude mixtures of about 30 to 70 percent by volume dehydrated alcoholfor each 30 to 70 percent by volume PEG 300 and/or PEG 400, which can bediluted with saline or dextrose infusion fluids for IV administration,and may be applied for use in administering epothilone D to patients viaIV administration. In such formulations, it is preferred that the amountof ethanol be minimized to avoid side effects associated with ethanoladministration. Optimal ratios of solvents may be readily obtained byone skilled in the field.

Further preferred formulations specifically designed for administeringepothilone D and analogs are disclosed in U.S. Pat. No. 7,091,193 (alsopublished as U.S. Patent Application Publication No. 2005/0148543), toKosan (now BMS). This patent describes a formulation wherein epothiloneD and a hydroxypropyl-beta-cyclodextrin are combined in an alcohol-watersolution that is then lyophilized. Embodiments involve use of about 10mg epothilone D and about 0.4 g of hydroxypropyl-beta-cyclodextrincombined in a 60% tert-butanol-water solution that is then lyophilized(ingredients can be reduced proportionately for preparation ofindividual, lower dosages units, according to the current invention).The lyophilized active ingredient “cake” can then be reconstituted forIV administration with use of water, ethanol, and/or glycol, which mayinclude propylene glycol, polyethylene glycol 400, polyoxyethylenesorbitan monooleate (sold under the trade name TWEEN 80), and relatedoxygenated hydrocarbons. It is understood that glycols of various chainlengths and molecular weights (e.g., polyethylene glycol 1000, otherTWEEN compounds) may be used.

As a more specific example, a formulation that may be used to deliverepothilone D to a patient according to the invention may comprise about0 to 50% propylene glycol, about 1 to 10% TPGS, about 0.5 to 10%ethanol, about 0-90% water, and/or about 5 to 85% PEG such as PEG-400.More specifically, a formulation may comprise:

50% propylene glycol, 10% TPGS, 10% ethanol, 30% water; or10% propylene glycol, 40% PEG-400, 5% TPGS, 5% ethanol, 40% water; or85% PEG-400, 10% TPGS, 5% ethanol; or8.5% PEG-400, 1% TPGS, 0.5% ethanol, 90% water.

One preferred method of administering epothilone D according to theinvention involves oral administration. U.S. Pat. No. 6,576,651discloses methods for oral administration of epothilones with use of oneor more pharmaceutically acceptable acid-neutralizing buffers. However,a preferred method of administration would involve use of a tablet orcapsule, including a solid tablet or capsule or fluid or gelatin-filledcapsule. A solid tablet or capsule of epothilone D may be prepared withone or more enteric coatings. Enteric coatings have been used for manyyears to arrest the release of the drug from orally ingestible dosageforms. Depending upon the composition and/or thickness, the entericcoatings are resistant to stomach acid for required periods of timebefore they begin to disintegrate and permit slow release of the drug inthe lower stomach or upper part of the small intestines. Examples ofsome enteric coatings are disclosed in U.S. Pat. Nos. 6,224,910,5,225,202, 2,809,918, 3,835,221, 4,728,512 and 4,794,001, each of whichis incorporated herein by reference.

An enteric coated tablet directed to use of epothilone D is described inU.S. patent application Ser. No. 11/281,834, incorporated herein byreference, which may be used to formulate tablets of capsules ofepothilone to practice the invention. This formulation involves use ofan inactive base particle, such as a sugar bead, to which the activeingredient (i.e., epothilone D), is applied, which is then encapsulatedby an enteric coating polymer, and/or one or more subcoat layers. Thebeads are then included within a capsule. Enteric coatings for use informulating epothilone D tablets or capsules may include enteric coatingpolymers, such as, for example, hydroxypropyl methylcellulose phthalate,polyvinyl acetate phthalate, cellulose acetate phthalate, acrylic acidcopolymers, and methacrylic acid copolymers. One example of amethacrylic acid copolymer that may be used to form an enteric coatingis EUDRAGIT® L-30-D 55 aqueous copolymer dispersion, which comprises ananionic copolymer derived from methacrylic acid and ethyl acrylate witha ratio of free carboxyl groups to the ethyl ester groups ofapproximately 1:1, and a mean molecular weight of approximately 250,000,which is supplied as an aqueous dispersion containing 30 weight %solids. EUDRAGIT® L-30-D 55 aqueous copolymer dispersion is supplied byRohm-Pharma Co., Germany.

In preparing enteric coated beads to form capsules of epothilone D, itmay be desirable to include one or more subcoat layers that are situatedbetween the epothilone D core and the enteric coating to minimizecontact between those layers. For example, suitable materials to formthe subcoat layer include starch; gelatin; sugars such as sucrose,glucose, dextrose, molasses, and lactose; natural and synthetic gumssuch as acacia, sodium alginate, methyl cellulose,carboxymethylcellulose, and polyvinylpyrrolidone (PVP) polymers andcopolymers such as PVP-PVA copolymers; celluloses such asethylcellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; polyethylene glycol; and waxes. The subcoat layer may furthercomprise one or more plasticizers, such as polyethylene glycol,propylene glycol, triethyl citrate, triacitin, diethyl phthalate,tributyl sebecate, or combinations thereof.

The tablet or capsule of epothilone D optionally may comprise othermaterials such as flavoring agents, preservatives, or coloring agents asmay be necessary or desired.

An appropriate dosage of epothilone D can be determined by one of skillin the art, taking into consideration the findings described hereintogether with typical factors such as the body mass of the patient, thephysical condition of the patient, and so on. The dosage should containepothilone D in an amount that is effective for treating Tau-associateddiseases, including tauopathies such as AD. Generally, a range for thedosage of epothilone D administered for the treatment of Tau-associateddiseases (including tauopathies such as AD) is considered to be between0.0001-10 mg/m², more preferably between 0.001-5 mg/m². Other, morepreferred dosage ranges for PO and IV administration are set forth abovein the alternative embodiments section. The units mg/m², are usedherein, for purposes of comparison with the chemotherapeutic dosagespreviously administered with epothilones and their analogs. However, theunits mg/m² can be readily converted to mpk, considering the animalspecies receiving (or having received) the drug and the patient'sbodyweight and/or height. For example, for a human patient weighingabout 70 kg, the dose range of 0.0001-10 mg/m² converts to about0.00003-0.3 mpk. Further information concerning dose conversions can befound at www.rphworld.com/viewlink-25045.html, and in Freireich et al.,Cancer Chemother. Reports, 50(4):219 (1966).

The drug can be administered daily, weekly, or on an intermittent basis.For example, the drug can be administered for three weeks on, followedby one week off, or for two weeks on, followed by one week off, or underother dosing schedules as can be determined by one skilled in the field.The particular dose selected will depend upon the mode of administrationand dosing regime selected. One preferred schedule is a once daily oraldosing schedule. When longer periods of time are prescribed between eachapplication (typically the case for IV administration), each unit dosemay be larger than when daily dosages are provided.

Notably, the dose of epothilone D that was administered to patients fortreatment of cancer in certain Phase II clinical trials was 100 mg/m²administered as a 90 minute infusion given weekly for 3 of 4 weeks(i.e., on days 1, 8, and 15, every 4 weeks), following Phase I trialsinvolving dose escalations of from 9 to 150 mg/m² for each dose. Thedose of drug contemplated for treatment of AD is about ten-fold less,and more likely, about 100-fold less, and in another contemplatedembodiment, even more than 1000-fold less, than the therapeutic dose ofepothilone D that was administered for treatment of cancer patients inclinical Phase II trials, although the dosing schedule and mode ofadministration will influence the dose.

The present invention will be explained in further detail by way ofnon-limiting examples below, which make reference to the appendeddrawings. The following methods were used in the experiments describedin the examples that follow the description of the methods.

METHODS FOR EXPERIMENTALS Examples 1 Through 5

The creation of Tg4510, an aggressive Tau transgenic mouse line, wasrecently described (Santacruz et al., 2005; Berger et al., 2007). TheTg4510 line expressed Tau-P301L, a Tau mutant found in FTDP-17, usingthe calmodulin kinase II promoter. The Tg4510 line was unique in severalrespects:

1. High level of Tau expression (β-fold relative to mouse Tau);2. Restriction of Tau expression to the frontal-temporal lobes (therebyavoiding the motoric deficits that had characterized previous Tau linesthat expressed Tau in the spinal cord); and3. Rapid and extensive neurodegeneration (60% CA1 neurons were lost by5.5 months) preceded by cognitive deficits measurable at 4.5 months.

Drug Preparation for Tg4510 Study

Epothilone D (Compound I) was dosed intraperitoneally with a 26-gaugeneedle, in 10% ethanol, 90% water, 10 ml/kg at 0 (vehicle), 1 mpk, and10 mpk. A 10× stock solution was made in 100% ethanol, and diluted justbefore dosing. Mice were dosed in 3 cohorts and data were combined togive a final N of 12, 9, and 15 for the vehicle, 1 mpk, and 10 mpkgroups, respectively. Mice were dosed in a chemical fume hood.

Injection and Behavioral Testing Schedule for Tg4510 Study

Tg4510 mice were used in this study. These mice are awell-characterized, aggressive model of tauopathy that overexpress humanP301L mutant Tau in the forebrain (Santacruz et al., 2005; Berger etal., 2007). The mice are characterized by accumulations of abnormalforms of Tau, including tangles similar to those observed in AD brain,behavioral deficits, and eventually neuronal loss. At 9 weeks (+/−15days) of age, mice were acclimated to handling with a single mockinjection of phosphate buffered saline, performed within a chemical fumehood. The mice were then housed in cages kept within the chemical fumehood for 48 hours. Following the 48-hour period, the mice weretransferred to clean cages and brought to a behavioral suite fortesting.

The mice were then tested in a Morris water maze (MWM) for six days. Themice were distributed into treatment groups based on the results of thebehavioral analysis using the rank scores for probe trial 2 annuluscrossing index. Mice were 11 weeks (+/−15 days) of age at the start ofdosing and were dosed once weekly. A panel of neurological and physicalpropensity tests (Modified SHIRPA) were performed following the firstweek of dosing, including analysis of body position, tremor, coatappearance, gait, touch escape, positional passivity, limb grasping, andrighting reflex. Mice were additionally examined 48 hours after eachweekly dose for coat appearance, limb grasping, righting reflex and forany overt stereotyped behavior. No signs of overt toxicity, weight loss,or motor deficits were observed in the course of the study.

Mice were again tested in the MWM after the eighth dose (19 weeks ofage+/−15 days) for six days. After behavioral testing, dosing resumeduntil the animals were 5.5 months of age at the time of harvest. Animalswere housed and treated according to Institutional Care and Animal UseCommittee and National Institutes of Health standards.

Morris Water Maze Protocol

Mice were tested in Morris water maze (MWM) on two occasions, once priorto dosing, and once two months after dosing began. The second round ofwater maze testing was performed in another testing room. Mice wereacclimated to the experimental room for 2-3 days prior to testing. Themice were placed in a water maze of 1.5 m diameter, with a 16 cmdiameter platform placed 0.5-1.0 cm under the surface of the water. Thewater was made opaque with non-toxic white paint and the watertemperature was regulated between 22-25° C.

The mice were given 4 trials per day of up to 90 seconds each with a 10second rest period on the platform after each trial. If the mouse didnot find the platform within 90 seconds, the mouse was gently guided tothe platform and allowed to remain there for 10 seconds. The testingroom rooms each had large external cues to allow the mice to orient asthey learned the location of the platform. Mice were placed under a heatlamp to prevent hypothermia after each trial. The interval betweentrials ranged from 25 to 45 minutes. The mice were tracked using HVSImage Advanced Tracker VP200 software (Buckingham, UK) and the totaldistance traveled until reaching of the platform was determined.

Statistical analysis for acquisition path length from the five trialsinvolved a repeated measures analysis of variance. The statistical modelincluded “treatment” (0, 1 mpk, or 10 mpk of epothilone D (Compound I))as a between animal term, and the 5 trials as repeated measures on eachanimal. If the analysis indicated a significant effect of treatment, ora treatment-by-trial interaction, differences between the 1 mpk and 10mpk groups were compared to the vehicle group using Dunnett's test. Theprobe pathlengths in each quadrant, and number of platform crossings ineach quadrant, were analyzed using Dunnett's test. In all cases, 1 mpkand 10 mpk groups were compared to the vehicle group. All calculationswere done in SAS, version 9.1, under the Windows XP Professionaloperating system.

Acquisition training was performed for 5 consecutive days. A Probe trialwas performed 18 h after the last acquisition training on day 6. Duringthese 60 second trials, the platform was removed, and the distance thatthe mouse spent in the target quadrant and the number of crossings of aregion where the platform was previously located were measured. Swimspeed was monitored for all animals; drug treatment did not cause anychanges in swim speed consistent with the drug not affecting motorbehavior. Float time (swim speeds of <5 cm/sec) also did not varybetween treatment groups.

Tissue Harvesting

Mice were euthanized by cervical dislocation at 5. 5 months followed bydecapitation. Brains were immediately removed and divided down themidline into two hemispheres. The right hemisphere was placed into 20 mLof 4% paraformaldehyde (prepared fresh on the day of sacrifice) andstored overnight at 4° C. The following day, the brains were transferredto a tube containing 20 mL TBS (pH 7.4, 20 mM TRIS, 100 mM NaCl) andthen stored at 4° C. until processing. Right hemispheres were embeddedin paraffin, sectioned at 5 microns, and mounted on positively chargedglass slides. The slides were dried overnight in a 60° C. oven andstored at room temperature until stained. The left hemispheres werefrozen (within 2 minutes) on dry ice.

Gallyas Method

The Gallyas staining method was used to detect silver-positiveneurofibrillary tangles and dystrophic neurites. Paraffin-embedded thinsections (5 microns) mounted on glass slides were deparaffinized andrehydrated via serial incubation in xylene (two times for 10 minuteseach), 100% ethanol (two times for 10 minutes each), 95% MeOH/5% H₂O₂(30 minutes), 95% ethanol (two times for 5 minutes each), 80% ethanol(two times for 5 minutes each), 50% ethanol (two times for 5 minuteseach), and water (two times for 5 minutes each). The sections were thenplaced into 5% periodic acid for 5 minutes, washed in dH₂O two times for5 minutes each time, and placed in alkaline silver iodide solution(containing 1% silver nitrate) for 1 minute.

The sections were washed in 0.5% acetic acid for 10 minutes, placed infreshly prepared developer solution for 15 minutes, and washed again in0.5% acetic acid for 5 minutes. Following a rinse in deionized water,the sections were placed in 0.1% gold chloride for 5 minutes and rinsedagain in deionized water. The sections were incubated in 1% sodiumthiosulphate (hypo) for 5 minutes and then rinsed in tap water.Counterstain was performed in 0.1% nuclear fast red for 2 minutes. Thesections were then rinsed in tap water, dehydrated in graded series ofalcohol (95%, 100%, 100%) for 2 minutes, and cleared in 3 changes ofxylene, 10 dips each. Finally, Cytoseal 60 mounting medium and coverslips were added to the slides (Richard-Allan Scientific of Kalamazoo,Mich.). Statistics was performed using the non-parametric Kruskal-Wallistest, followed by Dunn's multiple comparison test using Graphpad Prism4. The same results were obtained with the parametric ANOVA followed byDunnett's post-hoc test.

Immunohistochemistry

Paraffin-embedded thin sections (5 microns) were deparaffinized andrehydrated to water in 3 changes of xylene, two changes of 100% ethanol,and 1 change of 95% ethanol, followed by rinsing in water. Antigenretrieval was performed by steaming the slides in 10 mM sodium citratebuffer, pH 6.0 for 30 minutes in a Black and Decker Steamer (Model #HS900) and then cooled for 30 minutes. Endogenous peroxidase activity isremoved by incubation in 0.6% hydrogen peroxide in 90% MeOH for 15minutes. After washing in TBS, slides are blocked in 10% normal goatserum in TBS for one hour. This is followed by incubation of the AT8phosphoTau antibody (Pierce Biotechnology Inc., Rockford, Ill., Goedertet al., 1995) diluted in the blocking solution overnight at 4° C. After3 washes in TBS, the slides are incubated with an anti-mouse IgGantibody for 1 hour at room temperature. After washing in TBS, thesignal is detected using a Vectastain ABC Elite Kit (Vector LabsBurlingame, Calif.) for 1 hour followed by detection using thediaminobenzadine reagent from Vector labs. Nuclei were counterstainedblue with hematoxylin, followed by dipping slides 2 times in Scott's tapwater substitute (Surgipath #02900, Richmond, Ill.) and then rinsing intap water. The sections were then dehydrated in graded series of alcohol(95%, 100%, 100%) then cleared in 3 changes of xylene. Cover slips andCytoseal 60 mounting medium were then added.

Stereology

Nissl stained slides were scanned and digitized using the AperioScanScope (Aperio Technologies, Inc., Vista, Calif.). Images of theentire brain section were captured at high resolution and stored asfiles within Spectrum (Aperio software). To process images, a region of4,000×4,000 pixels including the entire hippocampus was captured usingthe extract tool and saved as a JPEG file for importing into Metamorph(Molecular Devices, Sunnyvale, Calif.) for quantification of cell losswithin the CA1 and CA3 regions of the hippocampus. A modified version ofthe single section dissector method (Moller et al. 1990) was utilized toquantify cell loss because of its suitability for thin,paraffin-embedded tissue sections. To obtain relative numbers of cells,every fifth section was collected as the paraffin-embedded brains werecut sagitally between the Bregma and approximately 0.75 mm laterally.Three regions were drawn and counted per section using Metamorphsoftware. The same regions were used for every image and 5 sections werecounted per animal, 5 slides apart. Statistics were performed usingANOVA followed by Dunnett's post-hoc test.

Example 1

The design of the Tg4510 experiment with epothilone D (Compound I) asdescribed above is depicted in FIG. 1. In this experiment, mice weretested at 2.5 months in the MWM and assigned to one of three groups(N=12, 13, 16) such that the pre-treatment performance of each group wasdetermined to be similar. Starting at 2.5 months, mice were administereda weekly intraperitoneal (IP) injection of either vehicle alone orvehicle with 1 mpk or 10 mpk of epothilone D (Compound I). At 4.5months, the mice were again tested in the MWM to determine the effect oftreatment on cognitive performance. After 5.5 months, mice wereeuthanized and brains were collected for subsequent analysis.

In tumor xenograft experiments, investigators typically administerepothilone D (Compound I) intraperitoneally at 30 mpk every other dayfor 5 days, yielding a cumulative dose of 150 mpk. (Chou et al., 1998)Hence, treatment with 1 mpk epothilone D (Compound I) for 12 weeks, asdescribed herein, is considered to be about 100-fold below the oncologydose, with the treatment at 10 mpk being about 10-fold below the typicaloncology dose administered in this type of experiment. When mice weredosed once weekly intraperitoneally with 1 mpk and 10 mpk epothilone D(Compound I) for 2 or 6 months, no histopathological abnormalities wereobserved in multiple tissues, including liver, kidney, heart, testes,adrenal gland, bone marrow, peripheral nerve, stomach, and small andlarge intestines.

FIG. 2 shows the results of a MWM test of the Tg4510 mice at 2.5 months,prior to dosing with epothilone D (Compound I) or with vehicle. Therewere no statistically significant differences between the groups priorto dosing in acquisition or during probe trials, which was the basis forseparating animals into groups. In other words, FIG. 2 operates as acontrol in showing the pre-treatment performance of each group wassimilar.

The Tg4510 mice were then administered epothilone D (Compound I)) onceweekly intraperitoneally at 1 mpk, 10 mpk, and with vehicle, and the MWMtest was performed at 4.5 months, following this weekly dosing over 12weeks. The results which are reported in FIG. 3, revealed that micetreated with 1 mpk epothilone D (Compound I) were able to locate thehidden platform in the MWM more quickly (i.e., in a statisticallysignificant manner (p<0.01)), than could mice that were treated with thevehicle. The 10 mpk treatment group showed a trend toward improvement ascompared with the vehicle group. These findings show that treatment ofTg4510 mice with epothilone D (Compound I) led to statisticallysignificant improved cognitive function relative to vehicle treatment,and additionally, that the lower dose of 1 mpk generated improvedresults as compared with the higher dose (10 mpk). Notably, theinventors herein further confirmed that the exposure using this paradigmwas dose dependent based on separate experiments comparing 1 mpk and 10mpk doses in mice. For this reason, the reduced behavioral improvementin the 10 mpk group, relative to the 1 mpk group, was not due tounanticipated, reduced drug levels in the 10 mpk treated animals.

Example 2

FIG. 4 shows probe data 18 h after 5 days of training in the 4.5month-old Tg4510 mice dosed for 2 months with epothilone D (Compound I)at 1 mpk, 10 mpk, and with vehicle. In FIG. 4, “TQ” stands for targetquadrant, “AR” stands for adjacent right, “AL” stands for adjacent left,and “OP” stands for opposite quadrant. Two measures of performance,namely % pathlength (A) and number of platform crossings (B) in eachquadrant, are indicated in FIG. 4. A preference for the target quadrantindicates that the mouse remembered the location where the platform waslocated during the acquisition phase of the study. As can be seen fromthe data, the vehicle-treated mice performed at chance with similarresults for each of TQ, AR, AL, and OP, for both the pathlength (A) andplatform crossing (B) measures, and they did not show a quadrantpreference. However, the mice treated with 1 mpk (Compound I) showedstatistically significant differences in both measures as compared withthe vehicle group in memory, e.g., in recalling that the platform hadbeen located at the TQ. Additionally, the 10 mpk group showedsignificantly greater performance compared to the vehicle group in the %pathlength measure (A) but not when using the number of platformcrossings measure (B).

Example 3

To determine the effect of epothilone D (Compound I) on brain pathology,brain tissue was examined from a subset (N=5) of the Tg4510 mice fromthe preceding experiment. Previous studies had shown that Tg4510 micelost about 60% of their neurons in the CA1 region of the hippocampus at5.5 months (Santacruz et al. 2005). Thus, the present inventors firstexamined the number of the neurons in the CA1 region of the hippocampus,followed by examination of the CA3 region. FIG. 5 depicts neuronalcounts in the CA1 and CA3 regions of hippocampus in the mice at 5.5months following treatment with vehicle, 1 mpk of epothilone D (CompoundI), and 10 mpk of epothilone D (Compound I).

Surprisingly, as can be seen in FIG. 5, the Tg4510 mice treated with 1mpk epothilone D (Compound I) had substantially more CA1 neurons thanvehicle-treated animals. In fact, the difference between the micetreated with vehicle and the mice treated with 1 mpk of epothilone D(Compound I) shows that the 1 mpk of epothilone D (Compound I) preventedneuronal loss with a statistically significant difference from vehicle(p<0.01). The mice treated with 10 mpk of epothilone D (Compound I) hadCA1 neuronal levels that were intermediate between the vehicle-treatedmice and the mice treated with 1 mpk of epothilone D (Compound I). Theseresults are consistent with and reinforce the findings from thebehavioral studies of Examples 1 and 2, i.e., showing that the 100-foldlower dose (i.e., than the chemotherapeutic dosages administered intumor xenograft experiments) consistently produced significantlyimproved results in treating tauopathy.

A similar trend was also observed for the total cell counts of CA3regions of the hippocampus, with significant differences between the 1mpk and non-transgenic mice compared to vehicle treated mice. Theelevation in cell count at the CA3 region in the treated group was lesspronounced than in the CA1 region where there is more neurodegenerationat this age; however, these results show an impact on underlying diseasein multiple regions of the brain.

Example 4

The effect of treatment on phosphorylated Tau staining in the CA1 regionwas also examined. The AT8 antibody recognizes Tau that isphosphorylated on both the 202 and 205 residues. This form ofhyperphosphorylated Tau is greatly enriched in AD and other Tauopathypatient brains. (Goedert et al. 1995).

FIG. 6 shows AT8 phosphoTau staining of the Tg4510 mice treated withvehicle, 1 mpk epothilone D (Compound I), and 10 mpk epothilone D(Compound I) as described above. PhosphoTau staining is indicated indark black. Surprisingly, the mice treated with 1 mpk of epothilone D(Compound I), showed much less phosphoTau staining, particularly incomparison to the vehicle-treated mice. Mice treated with 10 mpk ofepothilone D (Compound I) showed intermediate levels of phosphoTaustaining

Example 5

The effect of treatment with epothilone D (Compound I) onneurofibrillary tangle formation in the cortex was examined by Gallyassilver staining FIG. 7A shows Gallyas silver staining forneurofibrillary tangles in the frontal cortex of the Tg4510 mice treatedwith vehicle, 1 mpk of epothilone D (Compound I), and 10 mpk ofepothilone D (Compound I) as described above. In FIG. 7A, silverstaining is in black (positive), and “NT” stands for non-transgenic,demonstrating some non-specific staining associated with blood vessels.As can be seen in FIG. 7A, the mice treated with 1 mpk of epothilone D(Compound I) had much lower levels of neurofibrillary tangles than didvehicle-treated mice; this is quantitated for all animals in the studyin FIG. 7B. A significant impact on underlying disease in both cortexand hippocampous was observed at the 1 mpk dose, with the 10 mpk doseagain showing a trend toward improvement.

As described in the preceding Examples, treatment of Tg4510 mice withepothilone D (Compound I) prevented cognitive decline and improvedcognitive function over time as compared with the untreated Tg4510 mice.Furthermore, neuropathological tests as measures of impact on underlyingdisease (i.e., cell count, phosphoTau staining, and silver stainingtests), demonstrate that treatment with epothilone D prevents neuronalloss, reduces accumulation of abnormal Tau, and prevents the formationof neurofibrillary tangles at statistically significant levels ascompared with untreated Tg4510 mice. Thus, the inventors herein believethey are the first to discover and demonstrate the prevention ofcognitive loss, Tau pathology, and neurodegeneration upon treatment witha microtubule-stabilizing compound, namely, epothilone D.

Additionally, the inventors herein have discovered that the therapeuticeffects achievable upon treatment with epothilone D is likelynon-linearly dose dependent. Specifically, consistent dose-dependentresults were repeatedly obtained in each of the behavioral andneuropathological studies reported, wherein at the lower dose (1 mpk)(about 100-fold less than the chemotherapeutic dose in tumor xenograftexperiments), a significantly-enhanced beneficial effect was obtained inall measures as compared with the vehicle, while the higher dose (10mpk), showed a trend toward effect with most measures and astatistically significant difference over vehicle in one measure of theMWM probe test.

Example 6 Epothilone D Performance Compared with OtherMicrotubule-Stabilizers in Bolus IV Experiments

In one group of experiments, ixabepilone (aza-epothilone B analog),Compound II (BMS 310705, 21-amino epothilone F), and epothilone D(Compound I) were evaluated and compared to paclitaxel after bolus IVadministration into the tail veins of nude mice at dosages of 1 to 12mpk with 3 mice/group. Each of the four compounds were dosed at 5 ml/kgusing 10% Cremophor, 10% ethanol, and 80% water containing 5% dextrose.To determine the relative brain penetrance of each compound, the plasma,brain, and liver levels of the compounds were measured at various timesafter a single dose using liquid chromatography with tandem massspectrometry (LC/MS/MS) after an organic phase extraction, as reportedin FIGS. 8A-8D and Table 1. Liver levels were not measured in thepaclitaxel treated mice.

FIG. 8A shows the concentration of Compound II in the plasma, brain, andliver of mice following IV administration at 1 mpk at various times.

FIG. 8B shows the concentration of ixabepilone in the plasma, brain, andliver of mice following IV administration at 12 mpk at various times.

FIG. 8C shows the concentration of paclitaxel in the plasma and brain ofmice following IV administration at 4 mpk at various times.

FIG. 8D shows the concentration of epothilone D (Compound I) in theplasma, brain, and liver of mice following IV administration at 5 mpk atvarious times.

The data showed that Compound II and ixabepilone had modest brain levelsrelative to peripheral tissue as measured by the ratio of thebrain-to-liver compound levels, particularly at later times after theinitial distribution and clearance of plasma drug. As expected,paclitaxel brain levels were low. In particular, paclitaxel brain levelsdid not exceed plasma drug levels for at least 24 h after dosing.Unexpectedly, epothilone D (Compound I) had the combined properties ofremarkably better brain penetration and selective retention than thecompounds tested in this experiment, as evidenced by high brain levelsthat exceeded liver levels at 6 and 24 h after dosing. This demonstratesunexpected retention of epothilone D (Compound I) in the target organ(brain) relative to the periphery, including the plasma and tissues,most notably the liver, which is a potential site of toxicity.

More specifically, Table 1 reports comparative brain penetration datafor four microtubule stabilizers—paclitaxel, Compound II (BMS 310705),ixabepilone, and epothilone D—after bolus IV dosing (varied mpk, asreported in the table) using nude mice, which data is also reflected inFIGS. 8A-8D. The brain-to-plasma ratio generally increases with timeafter dosing for each compound due to the rapid loss from the plasma andretention of the drug in the brain by binding to microtubules. Thebrain-to-plasma ratio may then fall for compounds where there is lessretention in the brain, such as is observed for Compound II showing adecrease between 6 and 24 h. Despite the change in brain-to-plasma ratiowith time, this ratio provides a measure of the intrinsic brainpenetration for a compound when data from short times after dosing(e.g., between 20-60 minutes following dosing) are compared. In theTables herein, brain-to-plasma and brain-to-liver ratios were calculatedby first calculating the ratios for individual animals, and thendetermining the mean of the ratios; the Tables herein report the meanvalues thus obtained.

TABLE 1 Plasma Brain Brain-to- Brain-to- Dose Time conc conc plasmaLiver Compound (mpk) (hr) (nM) (nM) Ratio Ratio Paclitaxel 4 1 447 470.10 NQ 6 43 16 0.37 NQ 24 12 15 1.25 NQ Compound II 1 6 3 6.8 2.1 0.0124 1 1.3 1.2 0.02 Ixabepilone 12 0.12 11,236 579 0.05 0.05 0.33 3057 4950.16 0.09 1 390 284 0.73 0.07 2 171 360 2.1 0.11 6 53 371 7.0 0.30 24 8236 30 1.2 Epothilone D 5 6 6 2794 470 149 24 1 2046 2046 1204

Looking at Table 1, paclitaxel is poorly brain penetrant as evidenced bya brain-to-plasma ratio of 0.1 at 1 hour after dosing; ixabepilone ismore brain penetrant than paclitaxel with a brain-to-plasma ratio of0.73 at 1 hour after dosing (Table 1). At times from 6-24 h afterdosing, the brain-to-plasma ratio is a reflection of both intrinsicbrain penetration and retention (half-life) in the brain. The data at 6and 24 h after dosing of epothilone D shows at least a 60-fold increasein brain-to-plasma ratio above ixabepilone, the compound with the nexthighest brain-to-plasma ratio in this group.

The brain-to-liver ratios not only provide a more singular measure ofbrain retention and half life, but also selective retention compared toperipheral tissues. This is valuable because the liver, chosen largelybecause it is well perfused and tends to have higher levels than manyother peripheral tissues, contains microtubules where the compound canbe retained, unlike the non-cellular plasma. In contrast to thebrain-to-plasma ratio where the optimal measurement time is in the 20-60minute range, it is preferable to compare the brain-to-liver ratios atlater times after dosing (e.g., 24 h or more), when the plasma levelshave significantly decreased, thereby allowing a more accurate measureof the drug that is specifically retained within brain and liver cells.A comparison of the brain-to-liver ratios shows that epothilone D ishighly, selectively retained in the brain relative to the liver. Forinstance, the 24 hour brain-to-liver ratio of epothilone D is 1204, aremarkably, much higher ratio as compared with the lower ratios forixabepilone (1.2) and Compound II (0.02) in the same set of experiments.

In a separate experiment, epothilone D plasma and brain concentrationswere evaluated for longer periods of time, i.e., up to 168 h, followingbolus IV administration, using a similar protocol as described above,but with middle-aged triple transgenic mice (Oddo et al. 2003), in thehands of different scientists. The results of this experiment arereported below in Table 2 and in FIG. 10.

TABLE 2 (EPOTHLONE D ONLY) Time Plasma Brain Brain/Plasma Brain/Liver(hr) conc (nM) conc (nM) Ratio Ratio 0.05 25,100 1127 0.04 0.42 0.172003 595 0.28 1.1 .33 549 529 0.86 0.38 1 325 422 1.2 0.42 3 70 468 6.10.30 6 43 141 3.3 0.24 16 0.8 210 265 NQ 24 0.9 82.7 89 NQ 96 <LLQ (0.6nM) 25.2 NQ NQ 168 <LLQ (0.6 nM) 16.3 NQ NQ

These data demonstrate the extended retention of epothilone D in braintissue to at least 168 h (7 days) after a single dose. The absolutebrain levels and ratios in Tables 1 and 2 for epothilone D vary; it isimportant to note that the experiments described in Tables 1 and 2 wereseparately performed at different times by different scientists withdifferent strains of mice. The inventors have observed that smalldifferences in IV injection time can alter the exact exposure profile,particularly the maximal plasma concentration, which will influence thebrain concentration, and further, that the IV injection time can differbetween scientists. For this reason, it is best to compare the resultswithin a single experiment. Despite this issue, the overall trends andrelative characteristics of the microtubule-stabilizers as compared witheach other are consistent, and these results show that epothilone D ishighly brain penetrant with substantially improved brain penetration andretention as compared with Compound II, ixabepilone, and paclitaxel. Forexample, even when engaging in a comparison of data obtained from twoseparate experiments, the brain-to-plasma ratio of epothilone D at 6 hafter dosing was 2046 in Table and 89 in Table 2, still markedly greaterthan the ratios at the same times for paclitaxel (0.37), and Compound II(2.1) in Table 1. Because the liver levels in the study described inTable 2 fell below the lower level of quantitation (LLQ of 49 nM in thisstudy) at 24 hr, a brain-to-liver ratio was not quantifiable (NQ) atthis time.

WO 03/074053 A1 broadly discloses the use of certain epothilones for thetreatment of brain diseases. According to that publication, plasma andbrain levels for three epothilones (not including epothilone D) weremeasured during the first 40 minutes following bolus IV administrationat 5 mpk. The brain and plasma concentration data reported in WO03/074053 A1 for what is identified therein as compound 1:4,8-dihydroxy-16-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-1-oxa-7-(1-propyl)-5,5,9,13-tetramethyl-cyclohexadec-13-ene-2,6-dione,and paclitaxel, are reproduced in Table 1 below. Data was reported in WO03/074053 according to the units, μg/ml, and in minutes; this data wasconverted to nM and is reported in Table 3 in nM and hr, for purposes ofcomparison. This data (per Table 3) was not independently confirmed bythe inventors herein but rather, it is reproduced based on the valuespresented in that publication (as converted to hr and nM). Additionally,it is noted stereoisomerism and/or a method of preparation are notreported for compound 1 within WO 03/074053, and a 13 E/Z mixture isreferenced (see page 13, line 15).

TABLE 3 Time Plasma Brain Brain-to-plasma Compound (hr) conc. (nM) conc.(nM) Ratio Compound III 0.17 1540 580 0.4 0.33 1150 1540 1.3 0.67 5801150 2 Paclitaxel 0.17 940 <LLQ NQ 0.33 700 <LLQ NQ 0.67 230 <LLQ NQ

Because the data was reported to only 40 minutes, only brain penetrationcan be assessed from this study by examining the brain-to-plasma ratio.Paclitaxel is presumed to have poor brain penetrance (consistent withthe data in Table 1) because the brain levels are below LLQ, althoughthe level of detection was not disclosed. Measures of brain retentionwhich need to be measured at least 24 h post-dosing, and selective brainpenetration by comparison with peripheral tissues were not discussed inWO 03/074053 A1.

Example 7 Epothilone D Performance Following Oral Administration

To further analyze epothilone D's performance in treating tauopathies,the compound was evaluated in two experiments involving administrationto C57BL/6 mice at 10 mpk and 35 mpk by oral gavage, respectively.Additionally, in these experiments, an isomer of4,8-dihydroxy-16-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-1-oxa-7-(1-propyl)-5,5,9,13-tetramethyl-cyclohexadec-13-ene-2,6-dione(Compound III herein), was also evaluated and a side-by-side comparisonmade as between epothilone D and Compound III. Below, we first reportthe experimental detail for preparation and isolation of Compound III,and then the biological, in vivo data is described.

General Experimental Information:

In the following procedures, all temperatures are given in degreesCelsius. ¹H-NMR spectra were run on a Bruker 500, 400, or 300 MHzinstrument and chemical shifts were reported in ppm (δ) with referenceto tetramethylsilane (6=0.0). All evaporations were carried out underreduced pressure. Unless otherwise stated, LC/MS analyses were carriedout on a Waters instrument using a Phenomenex-Luna 3.0×50 mm S 10reverse phase column employing a flow rate of 4 mL/min using a 0.1% TFAin MeOH/water gradient [0-100% in 3 min, with 4 min run time], and a UVdetector set at 220 nm or Phenomenex-Luna 3.0×50 mm 10 u reverse phasecolumn employing a flow rate of 5 mL/min using a 10 mM ammonium acetateacetonitrile/water gradient [5-95% in 3 min, with 4 min run time] and aUV detector set at 220 nm. Unless otherwise stated, purifications weredone on 40-63 mesh silica gel columns, or using a BIOTAGE® Horizonsystem, or using specified HPLC equipment and conditions.

Step 1:

To a solution of phosphonium salt A (prepared according to Nicolaou etal., J. Am. Chem. Soc., 119:7974-7991 (1997); 40.5 g, 57.9 mmol) in 300mL THF at 0° was added sodium bis(trimethylsilyl)amide (63.7 mL, 63.7mmol), and the solution was stirred for 5 min. A solution of(6S)-6-methyl-7-(tetrahydro-2H-pyran-2-yloxy)heptan-2-one (preparedaccording to U.S. Pat. No. 7,326,798; 14.54 g, 63.7 mmol) in 50 mL ofTHF was added rapidly, and the mixture was allowed to warm to RT over 16h. The reaction mixture was poured into saturated NH₄Cl, and extractedwith EtOAc (300 mL). The organic layer was washed with brine, dried overmagnesium sulfate, filtered, and evaporated in vacuo. The crude productwas purified on a silica gel column (EtOAc/hexane 0-10%) to yield 16 g(30.7 mmol, 53%) of Synthesis Intermediate-1. ¹H NMR (500 MHz, CDCl₃) δppm 6.90 (s, 1H), 6.43 (s, 1H). 5.20-5.05 (m, 1H), 4.6-4.5 (m, 1H),4.15-4.00 (m, 1H), 3.9-3.8 (m, 1H), 3.6-3.3 (m, 2H), 3.25-3.05 (m, 1H),2.70 (s, 3H), 2.35-2.15 (m, 2H), 2.05-1.90 (m, 5H), 1.90-1.75 (m, 1H),1.75-1.65 (m, 3H), 1.65-1.45 (m, 6H), 1.45-1.25 (m, 3H), 1.15-1.00 (m,1H), 1.00-0.80 (m, 12H), 0.05-−0.05 (dd, 6H). MS (LCMS) [M+H]=522.44,[M+Na]=544.42.

Step 2:

To a solution of Synthesis Intermediate-1 (16 g, 30.7 mmol) in 300 mL ofethanol at RT was added p-toluenesulfonic acid monohydrate (5.83 g, 30.7mmol). The mixture was stirred for 7 h, poured into saturated NaHCO₃ andextracted twice with methylene chloride (300 mL). The combined organiclayers were washed with brine and dried over magnesium sulfate. Afterfiltration and removal of the solvent, the crude material was purifiedusing a BIOTAGE® system (EtOAc/hexane, 10-45%) to yield 9.8 g (22.4mmol, 73%) of Synthesis Intermediate-2. ¹H NMR (500 MHz, CDCl₃) δ ppm6.92-6.90 (m, 1H), 6.45-6.40 (m, 1H), 5.15-5.00 (m, 1H), 4.10-4.00 (m,1H), 3.50-3.30 (m, 2H), 2.69 (s, 3H), 2.35-2.15 (m, 2H), 2.1-1.9 (m,5H), 1.75-1.50 (m, 5H), 1.50-1.25 (m, 3H), 1.10-0.75 (m, 13H),0.05-−0.05 (dd, 6H). MS (LCMS) [M+H]=438.29, [M+Na]=460.24.

Step 3:

To a solution of oxalyl chloride (2.94 mL, 33.6 mmol) in 100 mL ofmethylene chloride was added DMSO (4.88 mL, 68.8 mmol) slowly at −78°.After stirring 10 minutes, Synthesis Intermediate-2 (7 g, 15.99 mmol) in100 mL methylene chloride was added and stirring was continued for 30min. TEA (11.14 mL, 80 mmol) was added, and the mixture was allowed towarm to −10°. After saturated NaHCO₃ was added, the reaction mixture wasextracted twice with methylene chloride (100 mL). The combined organiclayers were washed with brine, dried over Na₂SO₄, filtered andevaporated to give the crude product as a yellow oil. Filtration througha short SiO₂ column, eluting with 15% EtOAc/hexane solvent, andconcentration in vacuo provided 7 g (16 mmol, 100%) of SynthesisIntermediate-3 as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ ppm 9.6-9.5(d, 1H), 6.9 (m, 1H). 6.4 (m, 1H), 5.2-5.1 (m, 1H), 4.1-4.0 (m, 1H), 2.7(s, 3H), 2.3-2.2 (m, 3H), 2.2-1.8 (m, 5H), 1.7-1.5 (m, 4H), 1.4-1.2 (m,3H), 1.1-1.0 (m, 3H), 0.87 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H).

Step 4:

The method of Klar et al. (Angew. Chem. Int. Ed., 45:7942-7948 (2006))was followed. To 200 mL of THF at −78° was added 70 mL of 0.5M freshlyprepared LDA (35 mmol), followed by 8.48 g (35 mmol) of(S)-2-(2,2-dimethyl-1,3-dioxan-4-yl)-2-methylheptan-3-one (Klar et al.,Synthesis, 2:301-305 (2005)). Stirring was continued for 30 min at −30°.After cooling to −78°, a solution of 1.0M ZnCl₂ (35.0 mL, 35 mmol) wasadded, and the resulting solution was stirred for 20 min. A solution ofSynthesis Intermediate-3 (7 g, 16.06 mmol) in 50 mL of THF was addedover 20 min. The mixture was stirred for an additional 8 h at −78°. Themixture was poured into saturated NH₄Cl and extracted twice with EtOAc(300 mL). The organic layers were washed with brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified using a SiO₂ column(EtOAc/hexane, 0-10%) to provide 7.5 g (11 mmol, 69%) of SynthesisIntermediate-4 as the first eluting and major aldol isomer. ¹H NMR (500MHz, CDCl₃) δ ppm 6.90 (m, 1H), 6.43 (m, 1H), 5.15-5.05 (m, 1H),4.15-4.00 (m, 2H), 4.00-3.80 (m, 2H), 3.50-3.40 (m, 1H), 3.30-3.20 (m,1H), 2.85-2.75 (m 1H), 2.69 (s, 3H), 2.35-2.15 (m, 2H), 2.10-1.90 (m,5H), 1.75-0.75 (m, 41H), 0.05-−0.05 (d, 6H). MS (LCMS) [M+H]=678.47,[M+Na]=700.44.

Step 5:

To a solution of Synthesis Intermediate-4 (8 g, 11.8 mmol) in 200 mL ofmethylene chloride at 0° was added 2,6-lutidine (6.87 mL, 59 mmol),followed by tert-butyldimethylsilyl trifluoromethanesulfonate (8.13 mL,35.4 mmol). The mixture was allowed to warm to RT over 16 h, poured intosaturated NaHCO₃ and extracted with methylene chloride. The organiclayers were washed with brine, dried over Na₂SO₄ and the solvents wereremoved in vacuo. The residue was purified on a SiO₂ column(EtOAc/hexane, 5-10%) to yield Synthesis Intermediate-5 (7.8 g, 9.84mmol, 83%). ¹H NMR (500 MHz, CDCl₃) δ ppm 6.90 (s, 1H), 6.44 (s, 1H),5.15-5.05 (m, 1H), 4.25-4.20 (m, 1H), 4.10-4.00 (m, 1H), 4.00-3.90 (m,1H), 3.90-3.75 (m, 1H), 3.75-3.70 (m, 1H), 3.10-3.00 (1H), 2.70 (s, 3H),2.35-2.15 (m, 2H), 2.00-1.85 (m, 5H), 1.70-0.75 (m, 50H), 0.10-−0.05 (m,12H). MS (LCMS) [M+H]=792.48, [M+Na]=814.44.

Step 6:

To a solution of Synthesis Intermediate-5 (6.8 g, 8.58 mmol) in 100 mLof ethanol at RT was added p-toluenesulfonic acid monohydrate (1.8 g,9.44 mmol). After stirring for 6 h, saturated NaHCO₃ was added and themixture was extracted with EtOAc. The organic layers were washed withbrine, dried over Na₂SO₄ and concentrated in vacuo. The reaction wasrepeated using 1 g of Synthesis Intermediate-5. The combined crudeproducts were purified using a BIOTAGE® system (EtOAc/hexane, 10-40%) toyield Synthesis Intermediate-6 (5 g, 6.65 mmol, 68%). ¹H NMR (500 MHz,CDCl₃) δ ppm 6.9 (s, 1H), 6.44 (s, 1H), 5.15-5.05 (m, 1H), 4.15-4.00 (m,1H), 4.00-3.90 (m, 1H), 3.90-3.80 (m, 1H), 3.75-3.65 (m, 1H), 3.65-3.55(m, 1H), 3.10-2.90 (m, 2H), 2.69 (s, 3H), 2.25-2.15 (m, 2H), 2.00-0.75(m, 50H), 0.10-−0.05 (m, 12H). MS (LCMS) [M+H]=752.42.

Step 7:

To a solution of Synthesis Intermediate-6 (5 g, 6.65 mmol) in 200 mL ofmethylene chloride at 0° was added 2,6-lutidine (7.7 mL, 66.5 mmol),followed by tert-butyldimethylsilyl trifluoromethanesulfonate (9.16 mL,39.9 mmol). The mixture was allowed to warm to RT over 16 h, then pouredinto saturated NaHCO₃ and extracted with methylene chloride. The organicsolvent was evaporated and the crude mixture was filtered through alayer of SiO₂ with EtOAc/hexane (10-20%) to provide SynthesisIntermediate-7 as an oil (6.9 g, 100%). ¹H NMR (500 MHz, CDCl₃) δ ppm6.9 (s, 1H), 6.44 (s, 1H), 5.20-5.05 (m, 1H), 4.10-4.00 (m, 1H),3.95-3.85 (m, 1H), 3.80-3.75 (m, 1H), 3.70-3.60 (m, 1H), 3.60-3.50 (m,1H), 3.10-3.00 (m, 1H), 2.69 (s, 3H), 2.25-2.15 (m, 2H), 2.00-0.75 (m,67H), 0.10-−0.05 (m, 24H).

Step 8:

To a solution of Synthesis Intermediate-7 (5 g, 5.1 mmol) in 80 mL ofmethylene chloride and 40 mL of MeOH at 0° was added(+/−)-camphor-10-sulfonic acid (1.18 g, 5.1 mmol). The mixture wasstirred for 6 h at 0°, poured into saturated NaHCO₃ and extracted withmethylene chloride. The organic layers were washed with brine, driedover Na₂SO₄ and concentrated to yield Synthesis Intermediate-8 as an oil(3.6 g, 4.15 mmol, 81%). ¹H NMR (500 MHz, CDCl₃) δ ppm 6.90 (s, 1H),6.44 (s, 1H), 5.2-5.1 (m, 1H), 4.1-4.0 (m, 2H), 3.85-3.75 (m, 1H),3.7-3.6 (m, 2H), 3.1-3.0 (m, 1H), 2.7 (s, 3H), 2.3-2.2 (m, 2H), 2.0-1.9(m, 6H), 1.7-1.5 (m, 5H), 1.5-1.3 (m, 3H), 1.3-1.1 (m, 6H), 1.1-1.0 (m,4H), 1.0-0.8 (m, 35H), 0.1-−0.1 (m, 18H). MS (LCMS) [M+H]=866.49.

Step 9:

To a solution of oxalyl chloride (0.8 mL, 9.14 mmol) in 40 mL ofmethylene chloride at −78° was added DMSO (1.24 mL, 17.5 mmol). Afterstirring for 10 min, a solution of Synthesis Intermediate-8 (3.6 g, 4.15mmol) in 40 mL of methylene chloride was added. After 30 min, TEA (3.76mL, 27.0 mmol) was added and the reaction mixture was allowed to warm to0° over 2 h. Saturated NaHCO₃ was added and the mixture was extractedwith methylene chloride. The organic layers were washed with brine,dried over Na₂SO₄ and concentrated in vacuo to provide SynthesisIntermediate-9 as an oil (3.6 g, 4.16 mmol, 100%). ¹H NMR (500 MHz,CDCl₃) δ ppm 9.77 (m, 1H), 6.9 (s, 1H), 6.44 (s, 1H), 5.3 (s, 1H),5.2-5.1 (m, 1H), 4.5-4.4 (m, 1H), 4.1-4.0 (m, 1H), 3.8-3.7 (m, 1H),3.1-3.0 (m, 1H), 2.7 (s, 3H), 2.6-2.1 (m, 4H), 2.0-1.9 (m, 4H), 1.7-1.5(m, 4H), 1.5-1.3 (m, 5H), 1.3-1.1 (m, 5H), 1.1-1.0 (m, 3H), 1.0-0.8 (m,35H), 0.1-−0.1 (m, 18H).

Step 10:

To a solution of Synthesis Intermediate-9 (3.6 g, 4.16 mmol) in 120 mLof t-BuOH and 85 mL of THF at 0° was added 30 mL of water,2-methylbut-1-ene (18.5 g, 264 mmol), sodium dihydrogenphosphate (1.6 g,13.2 mmol) and sodium chlorite (2.98 g, 26.4 mmol). After stirring 2 hat 0°, the mixture was poured into saturated Na₂S₂O₃ solution (100 mL)and extracted three times with EtOAc (300 mL). The combined organiclayers were washed with brine, dried over Na₂SO₄, and concentrated invacuo. The residue was purified using a BIOTAGE® system (EtOAc/hexane,10-50%) to give Synthesis Intermediate-10 as a colorless oil (2.8 g,3.18 mmol, 72%). ¹H NMR (500 MHz, CDCl₃) δ ppm 6.93 (s, 1H), 6.66 (s,0.5H), 6.46 (s, 0.5H), 5.25-5.0 (m, 1H), 4.4-4.3 (m, 1H), 4.2-4.0 (m,1H), 3.9-3.7 (m, 1H), 3.2-3.0 (m, 1H), 2.7 (d, 3H), 2.6-2.4 (m, 1H),2.4-2.0 (m, 3H), 2.0-1.0 (m, 23H), 1.0-0.8 (m, 35H), 0.1-−0.1 (m, 18H).MS (LCMS) [M+H]=881.53.

Step 11:

To a solution of Synthesis Intermediate-10 (2.8 g, 3.18 mmol) in 5 mL ofTHF at RT was added TBAF (42 mL, 1.0M). The mixture was stirred for 6 h,then poured into saturated NH₄Cl, and extracted twice with EtOAc (300mL). The combined organic layers were washed with HCl (1.0 N, 200 mL),saturated NaHCO₃ and brine, and dried over Na₂SO₄ to give SynthesisIntermediate-11 as a viscous oil (2.6 g, 3.3 mmol, 100%). ¹H NMR (500MHz, CDCl₃) δ ppm 6.9 (s, 1H), 6.6-6.5 (m, 1H), 5.2-5.1 (m, 1H), 4.4-4.3(m, 1H), 4.15-4.05 (m, 1H), 3.8-3.7 (m, 1H), 3.4-3.2 (m, 1H), 3.1-3.0(m, 1H), 2.8-2.7 (m, 2H), 2.66 (d, 3H), 2.5-2.4 (m, 1H), 2.4-2.3 (m,2H), 2.3-2.2 (m, 1H), 2.0-0.8 (m, 46H), 0.1-0.0 (m, 12H). MS (LCMS)[M+H)=766.3, [M−H₂O]=748.3.

Step 12:

To a solution of Synthesis Intermediate-11 (2.6 g, 3.3 mmol) in 27 mL ofTHF at RT was added TEA (2.36 mL, 17 mmol), followed by2,4,6-trichlorobenzoyl chloride (3.31 g, 13.57 mmol). The reactionmixture was stirred for 20 min, then diluted with 260 mL of toluene. Thetoluene solution was added slowly to a stirred mixture of DMAP (3.86 g,31.6 mmol) in 1400 mL toluene over 4 h, after which TLC indicatedcompletion. HCl (4.0N, 12.5 mL) was added, and the solvent was removedin vacuo. The residue was partially purified using a BIOTAGE® system(EtOAc/hexane, 0-10%), providing a mixture which contained a mono-silylproduct and the above mixture (13 E/Z isomers) including Compound III(1.1 g). MS (LCMS) (520.2, 634.2). This material was subjected todeprotection without further purification.

Step 13: Compound III

To a solution of the reaction mixture above (102 mg) at −20° was added 1mL of TFA/CH₂Cl₂ (20% v/v). The reaction mixture was transferred to anice bath and stirred for 1 h. The solvent was removed in vacuo, addingsmall portions of toluene then re-evaporating, which provided a whitesolid. The same reaction was repeated with 125 mg of the partiallypurified mixture. The two reaction residues were combined and purifiedon a SiO₂ column (EtOAc/hexane, 20-35%) which provided a white solid(180 mg). The white solid was taken up in 5 mL of MeOH, and purified byHPLC (Varian, Dynamax PDA-2 detector; Waters C18 column; A: water with0.05% TFA; B: acetonitrile with 0.05% TFA, isocratic). Two major peakswere collected (Peak 1, 73.4 mg, 38%; and Peak 2, 41.8 mg, 22%).

Peak 1 was determined to be the 13-Z (1-oxa numbering) isomer byobservation of NOE between the C-14 olefinic proton and the C-13 methyl.¹H NMR (500 MHz, CDCl₃) δ ppm 7.14 (s, 1H), 6.75 (s, 1H), 5.15-5.05 (m,1H), 5.05-5.00 (m, 1H), 4.45-4.35 (m, 1H), 3.65-3.55 (m, 1H), 3.35-3.25(m, 1H), 2.92 (s, 3H), 2.55-2.45 (m, 2H), 2.35-2.25 (m, 2H), 2.25-2.15(m, 1H), 2.00 (s, 3H), 1.90-1.80 (m, 1H), 1.80-1.65 (m, 5H), 1.60-1.45(m, 2H), 1.45-1.30 (m, 5H), 1.25-1.15 (m, 3H), 1.05-0.95 (m, 6H),0.90-0.85 (t, 3H). MS (LCMS) [M+H]=520.3.

Peak 2 was determined to be the 13-E isomer (Compound III for Example 7experiment, below) by absence of NOE between the C-14 olefinic protonand the C-13 methyl. ¹H NMR (500 MHz, CDCl₃) δ ppm 7.03 (s, 1H), 6.65(s, 1H), 5.3-5.2 (m, 1H), 5.05-5.00 (m, 1H), 4.45-4.40 (m, 1H),3.65-3.60 (m, 1H), 3.4-3.3 (m, 1H), 2.77 (s, 3H), 2.6-2.3 (m, 4H),2.15-2.05 (m, 1H), 1.99 (s, 3H), 1.95-1.85 (m, 1H), 1.8-1.7 (m, 2H),1.57 (s, 3H), 1.50-1.35 (m, 4H), 1.29 (s, 3H), 1.25-1.10 (m, 3H),1.0-0.9 (m, 6H), 0.9-0.8 (t, 3H). MS (LCMS) [M+H]=520.3.

Oral In-Vivo Studies with Epothilone D and Compound III

For each compound (epothilone D and Compound III, prepared and describedas per the experiment immediately above), three mice per group (10 mpkand 35 mpk) were dosed at 10 ml/kg using 85% PEG-400, 10% TPGS, and 5.0%ethanol. At various intervals after dosing, the plasma, brain, and livercompound levels were measured following tissue homogenization,extraction with acetonitrile, and liquid chromatography with tandem massspectrometry (LC/MS/MS). Results from the studies are summarized inTables 4 and 5 and also, the results of the 35 mpk study are reported inFIG. 9. Specifically, Table 4 reports the concentration of epothilone D(Compound 1) and Compound III in the brain after oral administration (10mpk) up to 24 h after dosing (for Compound III, to the extent stilldetectible given LLQ), and Table 5 reports and FIG. 9 plots, theconcentration of epothilone D (Compound 1) and Compound III in the brainafter oral administration (35 mpk) up to 5 to 24 h after dosing (again,for Compound III, to the extent detectible). (A plot was not preparedfor the Table 4 data as only one brain concentration value wasdetectible for Compound III.) In Tables 4 and 5, below, where the valueswere <LLQ, the LLQ value is noted in the parenthetical.

TABLE 4 Brain/ Brain/ Time Plasma Brain Plasma Liver Compound (hr) conc(nM) conc (nM) Ratio Ratio Compound III 1 12.3 9.6 0.8 0.1 5 1.3 <LLQ NQNQ (3.7 nM) 7 1.2 <LLQ NQ NQ (3.7 nM) 24 <LLQ <LLQ NQ NQ (0.2 nM) (3.7nM) Epothilone D 1 15.1 10.6 0.7 0.4 3 3.9 7.0 1.8 1.3 4 2.5 9.9 4.0 3.48 1.4 6.4 4.6 1.8 13 0.1 6.3 47.3 5.1 24 0.2 9.1 44.5 8.0 48 <LLQ 5.4 NQNQ (0.1 nM) 96 <LLQ 3.0 NQ NQ (0.1 nM)

TABLE 5 Brain/ Brain/ Time Plasma Brain Plasma Liver Compound (hr) conc(nM) conc (nM) Ratio Ratio Compound III 1 47.7 67.7 2.3 0.4 5 3.0 4.61.4 NQ 24 0.7 <LLQ NQ NQ Epothilone D 1 52.7 61.3 1.1 0.5 5 3.6 76.925.2 11.3 24 <LLQ 118 NQ 18.7 (0.5 nM)

As can be seen, for Compound III, tissue levels from later times (i.e.,after 1 h or more) show that Compound III levels decreased rapidly inbrain tissue. Hence, Compound III has poor brain retention as observedin the lack of measurable brain levels at 24 h in both experiments. Oraldosing with epothilone D revealed brain-to-plasma ratios of 0.7 and 1.1at 1 hour, reflecting good brain penetrance. Unlike Compound III, brainlevels of epothilone D were maintained for more than 24 h (Tables 2, 4and 5, FIGS. 9-10). The brain-to-liver ratio for oral dosing ofepothilone D indicates that epothilone D is selectively retained in thebrain, consistent with the data in Tables 1 and 2 after IV dosing. Inparticular, the brain-to-liver ratio for epothilone D was 8 and 19 at 24h after dosing at 10 mpk and 35 mpk, respectively (Tables 4 and 5).These values reflect remarkably high selective brain-to-liver retentionrates for epothilone D.

Example 8 Epothilone D Half-Life Data Following IV, Oral, and IPAdministration

To further evaluate epothilone D's properties for treating tauopathies,the brain half-life of epothilone D was calculated from multiplestudies, and the results are reported in Table 6. To calculate anaccurate brain half-life for a long half-life compound, measurementsneed to be taken for several half lives after a single dose. From thestudy described in Table 2, where brain concentrations were measuredthrough 7 days after a single dose, the brain half life of epothilone D(Compound I) after IV dosing is 61 h (Table 6). The brain half life inmice after multiple routes of administration and dosages averaged46.0+/−7 h (Table 6). Similarly, the brain half-life after IV dosing inrats was 31 h (Table 6). In contrast, the brain half life of CompoundIII was clearly significantly shorter than epothilone D, as reflected inFIG. 9. As a further illustration of the epothilone D brain half-life,FIG. 10 is provided which plots the results of a study (data reported inTable 2, above), showing brain concentration levels at time periods ofup to 175 h post-dosing, following a 5 mpk bolus IV administration.

TABLE 6 Dose Half life Species Route (mpk) (hours) Mouse IV 5 61 MouseOral 10 46 Mouse IP 1 44 IP 10 41, 37, 45, 46 Rat IV 1 31

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We claim:
 1. A method of treating Alzheimer's Disease comprisingadministering a therapeutically-effective amount of epothilone D to apatient in need of treatment thereof.
 2. The method of claim 1, whereinthe epothilone D is administered orally.
 3. The method of claim 1,wherein the epothilone D is administered intravenously.
 4. The method ofclaim 1, wherein the epothilone D has brain penetrance of 0.5 or moremeasured at a period between 20 minutes and 1 hour post-dosing, andeither or both of 1) a brain half-life of 24 hours or more, and 2) brainto liver selective retention of 2 or more at 24 or more hourspost-dosing.
 5. The method of claim 1, wherein the method istherapeutically effective in treating Alzheimer's Disease in the patientwithout causing drug-induced side effects that would require that use ofthe epothilone D treatment be discontinued.
 6. The method of claim 1,wherein the cumulative monthly dose of epothilone D administered to thepatient is from about 0.001 mg/m² to about 30 mg/m².
 7. The method ofclaim 1, wherein the cumulative monthly dose of epothilone Dadministered to the patient is from about 0.001 mg/m² to about 6 mg/m².8. The method of claim 1, wherein the cumulative monthly dose ofepothilone D administered to the patient is from about 0.001 mg/m² toabout 3 mg/m².
 9. The method of claim 1, wherein the epothilone D isadministered intravenously to a human patient and the dose of epothiloneD administered over a one month cycle, regardless of schedule, is in therange of about 0.01 to about 5 mg/m².
 10. A method of treatingAlzheimer's Disease in a human patient, comprising the step ofadministering a therapeutically effective amount of epothilone D to thepatient, wherein the epothilone D is administered according to a dosingregime that provides a cumulative monthly dose of epothilone D to thepatient of from about 0.001 mg/m² to about 30 mg/m² and wherein theepothilone D is therapeutically effective in having an impact onunderlying disease and/or providing cognitive benefits to the patientwithout causing drug-induced side effects that would require that use ofthe epothilone D treatment be discontinued.
 11. The method of claim 10,wherein the cumulative monthly dose of epothilone D administered to thepatient is from about 0.001 mg/m² to about 6 mg/m².
 12. The method ofclaim 10, wherein the cumulative monthly dose of epothilone Dadministered to the patient is from about 0.001 mg/m² to about 3 mg/m².13. The method of claim 10, wherein the epothilone D is administeredintravenously to the human patient and the dose of epothilone Dadministered over a one month cycle, regardless of schedule, is in therange of about 0.01 to about 5 mg/m².